Recombinant yarrowia lipolytica with high yield of beta-caryophyllene as well as construction method and application thereof

Abstract

The invention belongs to the field of bioengineering, and provides recombinant yarrowia lipolytica with high yield of beta-caryophyllene as well as a construction method and application of the recombinant yarrowia lipolytica. The recombinant yarrowia lipolytica with high yield of beta-caryophyllene is named as a yarrowia lipolytica XJ-CAP strain, the recombinant yarrowia lipolytica XJ-CAP strain can be used for producing 6.27 g / L of beta-caryophyllene after being subjected to fed-batch fermentation, and the efficient synthesis of a plant source natural product beta-caryophyllene in the yarrowia lipolytica is realized. The construction method of the recombinant yarrowia lipolytica is easy to operate, and efficient synthesis of beta-caryophyllene can be achieved.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a recombinant Yarrowia lipolytica capable of producing high-yield beta-caryophyllene, a construction method and application thereof. Background Art

[0002] β-Caryophyllene (β-CAP) is mainly found in natural plant oils such as clove leaf oil, clove stem oil and cinnamon leaf oil. It is a bicyclic sesquiterpenoid compound mainly found in plants and can be used to synthesize flavor raw materials such as acetylcaryophyllene and other compounds. According to my country's GB 2670-1996 standard, β-caryophyllene is approved as a usable edible flavoring, especially in the formulation of spicy daily chemicals, edible flavors and tobacco flavors. It has a significant flavoring effect and can effectively coordinate the aroma and enhance the sweet smell. In addition, in the pharmaceutical industry, β-caryophyllene is used to develop anti-inflammatory drugs, analgesics and anti-cancer drugs due to its multiple biological functions such as antibacterial, anti-inflammatory and anti-depressant. In addition, β-caryophyllene also shows great potential as a potential new energy fuel, especially in the field of aviation fuel.

[0003] Traditionally, β-caryophyllene is mainly obtained by direct extraction from plants, but this method faces problems such as large planting area, long plant growth cycle, and susceptibility to pests and diseases and climatic conditions. In comparison, the microbial fermentation method has a short growth cycle and can be produced around the clock, so the use of microorganisms to ferment and produce β-caryophyllene is an economical and efficient way. In the existing technology, it is necessary to further improve the yield of heterologous synthesis of β-caryophyllene by transforming microorganisms through synthetic biology. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a recombinant Yarrowia lipolytica with high yield of β-caryophyllene and a construction method and application thereof in view of the deficiencies in the prior art.

[0005] In order to solve the above technical problems, the present invention discloses a recombinant Yarrowia lipolytica with high β-caryophyllene production and a construction method and application thereof. The following technical solutions are adopted:

[0006] A recombinant Yarrowia lipolytica with high beta-caryophyllene production, wherein the recombinant Yarrowia lipolytica expresses a heterologous gene; the heterologous gene is a gene encoding beta-caryophyllene synthase (AaCps, GenBank No. AGR40502.1) or an E353D mutant thereof from Artemisia annua; the gene encoding the beta-caryophyllene synthase or the E353D mutant thereof is obtained after codon optimization.

[0007] Among them, the nucleotide sequence of the β-caryophyllene synthase encoding gene derived from Artemisia annua is shown as SEQ ID No.1, and the nucleotide sequence of the β-caryophyllene synthase E353D mutant encoding gene derived from Artemisia annua is shown as SEQ ID No.2.

[0008] Wherein, the recombinant Yarrowia lipolytica expresses 1-3 copies of heterologous genes; preferably, the recombinant Yarrowia lipolytica expresses 2 copies of the gene encoding the β-caryophyllene synthase E353D mutant from Artemisia annua.

[0009] Wherein, the recombinant Yarrowia lipolytica simultaneously expresses any one or more combinations of 3-hydroxy-3-methylglutaryl CoA reductase encoding gene tHMG1, isopentenyl pyrophosphate isomerase encoding gene IDI and farnesyl pyrophosphate synthase encoding gene ERG20. Wherein, the GenBank accession number of the tHMG1 is XP_503558.1, the GenBank accession number of the IDI is YALI0F04015g, and the GenBank accession number of the ERG20 is YALI0E05753g. For detailed information, see Chinese patent CN116656518A.

[0010] The recombinant Yarrowia lipolytica simultaneously expresses the endogenous promoter P of the lipase encoding gene TGL4 TGL4 Replace with P YAT1 ; The P YAT1 The nucleotide sequence is shown in SEQ ID No. 3. Using the nitrogen transporter promoter P activated by the lipid phase endogenous Yarrowia lipolytica YAT1 Replace the native promoter (P TGL4 , 500 bp before ATG). TGL4 The nucleotide sequence is shown in SEQ ID No.4.

[0011] Preferably, the recombinant Yarrowia lipolytica further expresses one or more marker genes; the marker genes are selected from a 3(β)-isopropylmalate dehydrogenase encoding gene expression cassette or an orotidine-5'-phosphate decarboxylase encoding gene expression cassette.

[0012] Preferably, the recombinant Yarrowia lipolytica has KU70 gene knocked out. The starting strain of the recombinant Yarrowia lipolytica is Yarrowia lipolytica Po1f with KU70 gene knocked out, wherein Po1f was purchased from the American Type Culture Collection and is numbered ATCC MYA-2613.

[0013] Preferably, the recombinant Yarrowia lipolytica expresses two copies of the gene encoding the β-caryophyllene synthase E353D mutant from Artemisia annua, simultaneously expresses tHMG1, IDI and ERG20, and the endogenous promoter P of TGL4 is TGL4 Replace with P YAT1 ;

[0014] The nucleotide sequence of the gene encoding the β-caryophyllene synthase E353D mutant from Artemisia annua is shown in SEQ ID No. 2, the GenBank accession number of tHMG1 is XP_503558.1, the GenBank accession number of IDI is YALI0F04015g, the GenBank accession number of ERG20 is YALI0E05753g, and the P YAT1 The nucleotide sequence is shown in SEQ ID No.3.

[0015] In a second aspect, the present invention provides a method for constructing the recombinant Yarrowia lipolytica described in the first aspect, wherein the coding gene of the β-caryophyllene synthase or its E353D mutant is integrated into the yeast genome in the form of a gene expression cassette. Specifically, the method comprises the following steps:

[0016] (1) Constructing a heterologous gene expression cassette: amplifying a promoter, a heterologous gene and a terminator to obtain a promoter gene fragment, a heterologous gene fragment and a terminator gene fragment respectively, and connecting them to a plasmid vector at the same time to obtain a recombinant plasmid vector containing a heterologous gene expression cassette; preferably, the integration is in the Yarrowia lipolytica genome, and the integration site is any one or more of the A08 site, 26s rDNA site, IntA site, IntB site, IntC site, IntD site, IntE site, IntF site, lip1 site, SCP2 site, or YISCD site of Yarrowia lipolytica. The promoter is the promoter P of Yarrowia lipolytica. TEF , P hp4d , P TEFin , P FBA , P FBAin , P POX2 , P POT1 or P GPD Any one of; the terminator is the terminator T of Yarrowia lipolytica xpr2t , T mig1t , T lip2t , T cyc1t , T pex3t , T pex10t or T pex20tFurther preferably, the integration site is the A08 site and the IntB site of Yarrowia lipolytica, and the promoter is the promoter P of Yarrowia lipolytica TEFin , the terminator is the terminator T of Yarrowia lipolytica xpr2t .

[0017] (2) introducing the recombinant plasmid vector containing the heterologous gene expression cassette into Yarrowia lipolytica, so that the gene expression cassette is integrated into the Yarrowia lipolytica genome;

[0018] Preferably, the construction method comprises step (3), wherein the step (3) is to construct any one or more of a tHMG1 expression cassette, an IDI expression cassette, an ERG20 expression cassette and a promoter replacement expression cassette, and integrate them into the genome of the Yarrowia lipolytica constructed in step (2). The construction process and detailed information of the tHMG1 expression cassette, the IDI expression cassette and the ERG20 expression cassette can be found in Chinese patent CN116656518A.

[0019] Wherein, in step (3), the promoter replacement expression cassette comprises the upstream homology arm of the lipase gene TGL4 promoter, the promoter P YAT1 and the homology arm downstream of the start codon of the lipase encoding gene TGL4. Preferably, the promoter of the lipase encoding gene TGL4 is 500 bp before the start codon of the TGL4 gene.

[0020] The recombinant Yarrowia lipolytica constructed in steps (1)-(3) was named XJ-CAP strain.

[0021] In a third aspect, the present invention provides use of the recombinant Yarrowia lipolytica described in the first aspect in fermentation production of β-caryophyllene.

[0022] Preferably, the fermentation comprises the following steps: (1) culturing the recombinant Yarrowia lipolytica described in the first aspect using a fermentation medium without adding an organic solvent to obtain a fermentation product; (2) collecting the upper liquid by centrifugation, adding an organic phase, and centrifuging to obtain an organic phase rich in β-caryophyllene, wherein the organic phase is preferably n-dodecane.

[0023] Beneficial effects:

[0024] The recombinant Yarrowia lipolytica XJ-CAP strain constructed by the present invention is based on the Yarrowia lipolytica in which the non-homologous recombination gene KU70 encoding is knocked out, so that its homologous recombination ability is enhanced, and the integration of genes is achieved through the homologous recombination function of the Yarrowia lipolytica itself, which can greatly improve the genetic stability of the introduced genes. The recombinant Yarrowia lipolytica construction method is efficient and easy to operate.

[0025] Compared with the prior art, the recombinant Yarrowia lipolytica XJ-CAP strain of the present invention overexpresses two copies of the β-caryophyllene synthase E353D mutant encoding gene, the 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding gene, the farnesyl pyrophosphate synthase encoding gene, and the isopentenyl pyrophosphate isomerase encoding gene, and simultaneously utilizes the nitrogen transporter promoter P activated by the lipid phase endogenous Yarrowia lipolytica YAT1 By replacing the natural promoter of the endogenous lipase 4 encoding gene, experiments have shown that the recombinant Yarrowia lipolytica can produce 6.27 g / L of β-caryophyllene after fed-batch fermentation, realizing the efficient synthesis of the plant-derived natural product β-caryophyllene in Yarrowia lipolytica. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0027] Figure 1 This is a diagram of the β-caryophyllene synthesis metabolism in the present invention. Acetyl-CoA, acetyl-CoA; Acetoacetyl-CoA, acetoacetyl-CoA; ERG10, acetyl-CoA thiolase; ERG13, hydroxymethylglutaryl-CoA synthetase; HMGR, hydroxymethylglutaryl-CoA reductase; ERG12, mevalonate kinase; ERG8, mevalonate phosphate kinase; ERG19, mevalonate phosphate decarboxylase; IDI, isopentenyl pyrophosphate isomerase; ERG20, farnesyl pyrophosphate synthetase; AaCPS, Artemisia annua annua-derived β-caryophyllene synthase; TGL4, lipase 4; β-O, β-oxidation pathway; LB, lipid droplet; β-CAP, β-caryophyllene; HMG-CoA, hydroxymethylglutaryl-CoA; Mevalonate, mevalonate; Mevalonate-P, 5-mevalonate phosphate; Mevalonate-PP, 5-mevalonate pyrophosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; MVA Pathway, mevalonate pathway.

[0028] Figure 2 is the plasmid map of the recombinant plasmid pUC-A08-AaCPS, where A08-up represents the upstream homology arm of the A08 site, A08-dm represents the downstream homology arm of the A08 site, and TEFin represents the promoter P TEFin , xpr2t indicates the terminator T xpr2tleu represents the 3(β)-isopropylmalate dehydrogenase encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , 3(β)-isopropylmalate dehydrogenase and terminator T xpr2t ), AaCPS is the gene encoding β-caryophyllene synthase.

[0029] Figure 3 is the plasmid map of the recombinant plasmid pUC-HUH-PTGL4-PYAT1, wherein TGL4-up represents the 1500 bp homology arm upstream of the 500 bp position (promoter) of the TGL4 start codon, TGL4-dm represents the 1500 bp homology arm downstream of the TGL4 start codon, PYAT1 represents the lipid phase activated nitrogen transporter promoter endogenous to Yarrowia lipolytica, and URA represents the orotidine-5'-phosphate decarboxylase encoding gene expression cassette (containing the endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ).

[0030] Figure 4 Comparison of the content of β-caryophyllene produced by fermentation of recombinant strains 1 to 8 (YL-1 to YL-8). CAP: β-caryophyllene. DETAILED DESCRIPTION

[0031] The present invention is further described below by specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] In the following examples, the YPD liquid culture medium has a formula of: 20 g / L peptone, 10 g / L yeast extract and 20 g / L glucose.

[0033] The YPD plate containing 5-fluoroorotic acid has a formula of: dissolving 1 g of 5-fluoroorotic acid powder in 10 mL of dimethyl sulfoxide (DMSO) to obtain a 5-fluoroorotic acid solution. Adding 1 volume portion of the 5-fluoroorotic acid solution to 99 volume portions of YPD solid culture medium (adding 23 g / L agar powder to YPD liquid culture medium) to obtain a YPD plate containing 5-fluoroorotic acid.

[0034] The formula of the screening medium SD-Leu is: glucose 20g / L, YNB (amino-free yeast nitrogen source, purchased from BBI Life Sciences) 6.7g / L, CSM-Leu (complete supplement mixture minus leucine, purchased from MP Biomedicals) 0.67g / L, agar powder 23g / L, and the solvent is water.

[0035] The formula of the screening medium SD-Ura is: glucose 20 g / L, YNB (amino-free yeast nitrogen source, purchased from BBI LifeSciences) 6.7 g / L, CSM-Ura (complete supplement mixture minus uracil, purchased from MP Biomedicals) 0.67 g / L, agar powder 23 g / L, and the solvent is water.

[0036] Yarrowia lipolytica Po1f was purchased from the American Type Culture Collection and is numbered ATCCMYA-2613. Yarrowia lipolytica Po1fΔku70 (MatA, Δku70::HisG, leu2-270, ura3-302, xpr2-322, axp1-2), abbreviated as Yarrowia lipolytica Po1fΔku70. Yarrowialipolytica Po1fΔku70 was constructed by knocking out the coding gene KU70 responsible for non-homologous recombination from Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1-2): 63-72).

[0037] The A08 site integration plasmid is obtained by inserting a 2521 bp sequence (upstream homology arm) upstream of the A08 site start codon on the chromosome in the Yarrowia lipolytica Po1fΔku70 genome and a 2031 bp sequence (downstream homology arm) downstream of the stop codon into a pUC57-Leu vector (the construction method is shown in Example 1), and the Leu encoding gene is between the upstream and downstream homology arms of the A08 site.

[0038] The IntB site integration plasmid is obtained by inserting a sequence of 1500 bp upstream of the start codon of the IntB site on chromosome B in the Yarrowia lipolytica Po1fΔku70 genome (upstream homology arm) and a sequence of 1500 bp downstream of the stop codon (downstream homology arm) into a pUC57-HisG-Ura-HisG vector (the construction method is shown in Example 1), and two HisG tag encoding genes are located between the upstream and downstream homology arms of the IntB site.

[0039] In the following examples, the promoter P TEFin The nucleotide sequence is shown in SEQ ID No.5, and the terminator T xpr2t The nucleotide sequence is shown in SEQ ID No.6.

[0040] The metabolic pathway for synthesizing β-caryophyllene by introducing β-caryophyllene synthase or its E353D mutant from Artemisia annua of the present invention is as follows: Figure 1 shown.

[0041] Example 1 Amplification of genetic elements and preparation of target plasmids

[0042] (I) Preparation of target gene

[0043] According to the nucleotide sequence of the β-caryophyllene synthase encoding gene from Artemisia annua provided by NCBI (GenBank accession number: AGR40502.1), after codon optimization, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the optimized β-caryophyllene synthase encoding gene AaCPS (SEQ ID No. 1) and inserted into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-AaCPS.

[0044] According to the nucleotide sequence of the β-caryophyllene synthase E353D mutant encoding gene from Artemisia annua provided by NCBI, after codon optimization, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the optimized β-caryophyllene synthase E353D mutant encoding gene AaCPS-E353D (SEQ ID No. 2) and insert it into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-AaCPS-E353D.

[0045] According to the nucleotide sequence of the orotidine-5'-phosphate decarboxylase encoding gene URA of Yarrowia lipolytica provided by NCBI (GenBank accession number: AJ306421.1) and the HisG tag (GenBank accession number: AF324729.1), Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the two HisG tag encoding gene sequences into the plasmid pUC57, and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette (driven by the endogenous promoter P of Yarrowia lipolytica) was inserted between the two HisG tag encoding gene sequences. TEFin , orotidine-5'-phosphate decarboxylase encoding gene URA and terminator T xpr2t composition) to achieve Ura marker recovery and obtain plasmid pUC57-HisG-Ura-HisG (pUC57-HUH).

[0046] According to the nucleotide sequence of the 3(β)-isopropylmalate dehydrogenase encoding gene LEU of Yarrowia lipolytica provided by NCBI (GenBank accession number: M37309.1), Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize it. The expression cassette of the 3(β)-isopropylmalate dehydrogenase encoding gene LEU (driven by the endogenous promoter P of Yarrowia lipolytica) was inserted into the plasmid pUC57. TEFin , 3(β)-isopropylmalate dehydrogenase encoding gene Leu and terminator T xpr2t composition), and obtained plasmid pUC57-Leu.

[0047] (II) Construction of recombinant plasmid

[0048] The structure of the recombinant plasmid is shown in Table 1; the primers used to construct the recombinant plasmid are shown in Table 2.

[0049] 1. Construction of recombinant plasmid pUC-A08-AaCPS

[0050] The recombinant plasmid pUC-A08-AaCPS is based on pUC57-Leu, with the homology arm A08-up upstream of the start codon and the homology arm A08-dm downstream of the stop codon of the A08 locus in Yarrowia lipolytica Po1fΔku70 inserted, and the AaCPS expression cassette (P TEFin -AaCPS-T xpr2t ), LEU expression cassette (containing Yarrowia lipolytica endogenous promoter P TEFin , 3(β)-isopropylmalate dehydrogenase encoding gene LEU and terminator T xpr2t) is also between the upstream and downstream homology arms. For the specific structure, see Figure 2 .

[0051] Using Yarrowia lipolytica Po1fΔku70 genomic DNA as template, the AaCPS expression cassette promoter P TEFin and terminator T xpr2t .

[0052] The AaCPS gene was amplified using the primers in Table 2 using plasmid pUC57-AaCPS as a template.

[0053] The PCR amplification system is as follows: PrimerSTAR Max Premix 25 μL, template 1 μL, primer F 2 μL, primer R 2 μL, and distilled water 20 μL.

[0054] Among them, PrimerSTAR Max Premix was purchased from Baori Medical Biotechnology (Beijing) Co., Ltd.

[0055] The PCR program was as follows: denaturation at 98°C for 10 s, annealing at 55°C for 5 s, and extension at 72°C (extension time = target fragment length / 1 kb, unit: min), and repeated for 30 cycles.

[0056] Each fragment was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Bioshine Biotechnology Co., Ltd.).

[0057] The A08 site integration plasmid was digested with restriction endonuclease SnaBⅠ from NEB Company, and the linearized A08 site integration plasmid was recovered by agarose gel electrophoresis.

[0058] The linearized A08 site was integrated into the plasmid and the elements (promoter P TEFin , gene AaCPS and terminator T xpr2t ) One-step cloning was achieved using the ClonExpressMultiS One Step Cloning Kit from Nanjing Novogene Biotechnology Co., Ltd. The AaCPS gene expression cassette was inserted between the upstream and downstream homology arms of the A08 site integration plasmid to obtain a circular recombinant vector.

[0059] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened on LB plates for ampicillin resistance and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-A08-AaCPS.

[0060] The plasmid pUC-A08-AaCPS was digested with restriction endonuclease NotⅠ from NEB Company, and the linearized pUC-A08-AaCPS plasmid was recovered by agarose gel electrophoresis and used for yeast transformation.

[0061] 2. Construction of recombinant plasmid pUC-A08-AaCPS-E353D

[0062] The construction method is similar to that of the recombinant plasmid pUC-A08-AaCPS, except that the AaCPS expression cassette is replaced with the AaCPS-E353D expression cassette, that is, each element (promoter P TEFin , gene AaCPS-E353D and terminator T xpr2t ) The primers and templates used in amplification are different, see Table 2 for details.

[0063] 3. Construction of recombinant plasmid pUC-HUH-IntB-AaCPS-E353D

[0064] The recombinant plasmid pUC-HUH-IntB-AaCPS-E353D is based on pUC57-HisG-Ura-HisG, with the homology arm IntB-up upstream of the start codon and the homology arm IntB-dm downstream of the stop codon of the IntB site in Yarrowia lipolytica Po1fΔku70 inserted, and the AaCPS-E353D gene expression cassette (P TEFin -AaCPS-E353D-T xpr2t ), a URA expression cassette containing HisG fragments at both ends (containing the endogenous promoter P of Yarrowia lipolytica TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-A08-AaCPS. The primers described in Table 2 are used to amplify the expression cassette P using the recombinant plasmid pUC-A08-AaCPS-E353D as a template. TEFin -AaCPS-E353D-T xpr2t The IntB site integration plasmid was digested with the restriction endonuclease SnaBⅠ from NEB, and the linearized IntB site integration plasmid was recovered by agarose gel electrophoresis. TEFin -AaCPS-E353D-T xpr2t The linearized IntB site integration plasmid was cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntB-AaCPS-E353D.

[0065] 4. Construction of recombinant plasmid pUC-HUH-IntB-2AaCPS-E353D

[0066] The recombinant plasmid pUC-HUH-IntB-2AaCPS-E353D is based on pUC57-HisG-Ura-HisG, and is inserted with the homology arm IntB-up upstream of the start codon and the homology arm IntB-dm downstream of the stop codon of the IntB site in Yarrowia lipolytica Po1fΔku70. Two copies of the AaCPS-E353D gene expression cassette (P TEFin -AaCPS-E353D-T xpr2t ), a URA expression cassette containing HisG fragments at both ends (containing the endogenous promoter P of Yarrowialipolytica TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. The construction method is similar to that of the recombinant plasmid pUC-HUH-IntB-AaCPS-E353D. The primers (serial numbers 15 to 18) described in Table 2 are used to amplify the expression cassette 1-P using the recombinant plasmid pUC-A08-AaCPS-E353D as a template. TEFin -AaCPS-E353D-T xpr2t and 2-P TEFin -AaCPS-E353D-T xpr2t The IntB site integration plasmid was digested with restriction endonuclease EcoRⅠ from NEB, and the linearized IntB site integration plasmid was recovered by agarose gel electrophoresis. TEFin -AaCPS-E353D-T xpr2t , 2-P TEFin -AaCPS-E353D-T xpr2t The linearized IntB site integration plasmid was cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntB-2AaCPS-E353D containing two copies of AaCPS.

[0067] 5. Construction of recombinant plasmid pUC-HUH-PTGL4-PYAT1

[0068] The recombinant plasmid pUC-HUH-PTGL4-PYAT1 was based on pUC57-HisG-Ura-HisG, and inserted into the 500 bp before the start codon of TGL4 (GenBank accession number: NC_090775.1) in Yarrowia lipolytica Po1fΔku70 (i.e., the promoter position, TGL4 natural promoter P TGL4The nucleotide sequence of the nucleotide sequence is shown in SEQ ID No. 4) with a 1500 bp homology arm TGL4-up upstream and a 1500 bp homology arm TGL4-dm downstream of the TGL4 start codon, and a promoter P is inserted between the upstream and downstream homology arms. YAT1 , (P YAT1 The nucleotide sequence is shown in SEQ ID No. 3) and a URA expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t The construction method is similar to that of the recombinant plasmid pUC-A08-AaCPS. The upstream and downstream homology arms and the endogenous promoter P were amplified using the primers described in Table 2 and the genome of Yarrowia lipolytica Po1fΔku70 as a template. YAT1 The pUC57-HisG-Ura-HisG plasmid was double-digested with restriction endonucleases EcoRⅠ and HindⅢ from NEB, and the linearized pUC57-HisG-Ura-HisG plasmid was recovered by agarose gel electrophoresis. YAT1 The positive recombinant plasmid pUC-HUH-PTGL4-PYAT1 was obtained by one-step cloning and verification of the linearized pUC57-HisG-Ura-HisG plasmid. Figure 3 shown.

[0069] 6. Construction of recombinant plasmids pUC-HUH-IntC-tHMG1, pUC-HUH-IntF-IDI and pUC-HUH-SCP2-ERG20

[0070] The construction methods of the recombinant plasmids pUC-HUH-IntC-tHMG1, pUC-HUH-IntF-IDI and pUC-HUH-SCP2-ERG20 are detailed in Example 1 of patent CN116656518A.

[0071] Table 1 Insertion sequence information of each recombinant plasmid

[0072]

[0073]

[0074] Table 2 Primer sequences

[0075]

[0076]

[0077] Example 2 Construction of recombinant Yarrowia lipolytica (I) Construction of recombinant bacteria 1-2 (expressing AaCPS or AaCPS-E353D)

[0078] The recombinant plasmid pUC-A08-AaCPS was introduced into Yarrowia lipolytica Po1fΔku70, and the AaCPS expression cassette was integrated into the A08 site of the genome to obtain recombinant bacteria 1.

[0079] The recombinant plasmid pUC-A08-AaCPS-E353D was introduced into Yarrowia lipolytica Po1fΔku70, and the AaCPS-E353D expression cassette was integrated into the A08 site of the genome to obtain recombinant bacteria 2.

[0080] The specific method for constructing the recombinant bacteria 1 is as follows:

[0081] Yarrowia lipolytica Po1fΔku70 was cultured overnight in YPD liquid medium. Competent cells were prepared using Zymogen Frozen EZ Yeast Transformation Kit II from ZymoResearch Corporation, and the linearized recombinant plasmid pUC-A08-AaCPS was transformed into Yarrowia lipolytica Po1fΔku70 competent cells for homologous recombination. Positive clones were screened using screening medium SD-Leu, and then identified by PCR. The strain identified correctly by PCR was the recombinant strain 1.

[0082] The construction method of recombinant bacteria 2 was the same as that of recombinant bacteria 1, except that the linearized plasmid pUC-A08-AaCPS was replaced with the linearized plasmid pUC-A08-AaCPS-E353D.

[0083] (II) Construction of recombinant bacteria 3-4 (multi-copy expression of AaCPS-E353D)

[0084] The recombinant plasmid pUC-HUH-IntB-AaCPS-E353D was introduced into recombinant bacteria 2, and the AaCPS-E353D expression cassette was integrated into the genomic IntB site to obtain recombinant bacteria 3, which then lost a HisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure so that it could be used for the next round of Ura selection.

[0085] The recombinant plasmid pUC-HUH-IntB-2AaCPS-E353D was introduced into recombinant bacteria 2, and two copies of the AaCPS-E353D expression cassette were integrated into the genomic IntB site to obtain recombinant bacteria 4, which then lost a HisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure so that it could be used for the next round of Ura selection.

[0086] The specific method for constructing recombinant bacteria 3 is as follows:

[0087] (1) Yarrowia lipolytica Po1fΔku70 was cultured overnight in YPD liquid medium, and competent cells were prepared using Zymogen Frozen EZ Yeast Transformation Kit II from ZymoResearch Corporation. The linearized recombinant plasmid pUC-HUH-IntB-AaCPS-E353D was transformed into recombinant bacteria 2 for homologous recombination. Positive clones were screened using screening medium SD-Ura, and then identified by PCR.

[0088] (2) The positive clones identified by PCR were spread on a YPD plate containing 5-fluoroorotic acid, and then single colonies were streaked on both the YPD plate containing 5-fluoroorotic acid and the SD-Ura plate. Single colonies that could grow on the YPD plate containing 5-fluoroorotic acid but could not grow on the SD-Ura plate were selected to obtain recombinant bacteria 3.

[0089] The construction method of recombinant strain 4 is the same as that of recombinant strain 3, except that the plasmid used for homologous recombination needs to be changed accordingly. (IV) Construction of recombinant strains 5-7 (enhanced terpenoid precursor pathway, overexpression of tHMG1, IDI and ERG20)

[0090] The recombinant plasmid pUC-HUH-IntC-tHMG1 was introduced into the recombinant bacteria 3 that had lost Ura, and the tHMG1 expression cassette was integrated into the IntC site of the genome to obtain recombinant bacteria 5, which then lost a HisG tag and Ura screening marker under the 5-fluoroorotic acid selection pressure, so as to be used for the next round of Ura selection. pUC-HUH-IntF-IDI was introduced into the recombinant bacteria 5 that had lost Ura, and the IDI expression cassette was integrated into the IntF site of the genome to obtain recombinant bacteria 6, which then lost a HisG tag and Ura screening marker under the 5-fluoroorotic acid selection pressure, so as to be used for the next round of Ura selection. pUC-HUH-SCP2-ERG20 was introduced into the recombinant bacteria 6 that had lost Ura, and the ERG20 expression cassette was integrated into the SCP2 site of the genome to obtain recombinant bacteria 7, which then lost a HisG tag and Ura screening marker under the 5-fluoroorotic acid selection pressure, so as to be used for the next round of Ura selection.

[0091] The construction method of recombinant bacteria 5-7 is the same as that of recombinant bacteria 3, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0092] (V) Construction of recombinant bacteria 8

[0093] The recombinant plasmid pUC-HUH-PTGL4-PYAT1 was introduced into the recombinant bacteria 7 that had lost Ura, and the endogenous promoter P YAT1 Replace the natural promoter of lipase gene TGL4 TGL4 , and obtained the recombinant bacterium 8.

[0094] The construction method of recombinant bacteria 8 is the same as that of recombinant bacteria 3, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0095] Example 3 Application of recombinant Yarrowia lipolytica in the production of β-caryophyllene (I) Cultivation of engineered bacteria and product extraction

[0096] The initial bacteria Yarrowia lipolytica Po1f-Δku70 and the recombinant bacteria 1 to 8 (YL-1 to YL-8) in Example 2 were used to produce β-caryophyllene. The specific method is as follows: the initial bacteria and the recombinant bacteria were activated, and cultured in YPD liquid medium at 30°C and 220rpm for 16 hours to obtain seed liquid. The seed liquid was inoculated into 50mL fermentation medium YPD80 at an inoculum amount of 1% v / v, and cultured at 30°C and 220rpm for 5 days. After the fermentation was completed, the fermentation broth was transferred to a 50mL centrifuge tube, 10% v / v of n-dodecane was added, centrifuged at 5000rpm for 15min, and 1μL was taken for gas phase detection of β-caryophyllene concentration. The remaining sample was then desupernatanted and placed in an oven (75°C) to dry to constant weight for measuring biomass.

[0097] The formula of the fermentation medium YPD80 is: 20g / L peptone, 10g / L yeast extract and 80g / L glucose, and the solvent is water.

[0098] (II) Qualitative and quantitative analysis of β-caryophyllene

[0099] 1. β-Caryophyllene detection

[0100] Detection conditions: The β-caryophyllene content in the dodecane phase was measured by Agilent GC8860 using a DB-5ms column (30m×0.25mm×0.25μm) according to the following procedure: injection port 240℃, FID 240℃, split ratio 50:1, helium flow rate 3.0mL / min, 150℃ for 1 minute, increased to 280℃ at a rate of 20℃ / min, and maintained for 6 minutes. The sample injection volume was 1μL.

[0101] After 5 days of fermentation, no β-caryophyllene was detected in Yarrowia lipolytica Po1fΔku70, as Figure 4 As shown, the concentrations of β-caryophyllene (CAP) of recombinant bacteria 1 to 8 (YL-1 to YL-8) were 0.12, 0.26, 0.67, 0.51, 0.74, 0.79, 0.82 and 1.52 g / L, respectively. Among them, the β-caryophyllene yield of recombinant bacteria 8 was the highest, reaching 1.52 g / L, that is, 1.52 g of β-caryophyllene was produced per liter of fermentation liquid, which was significantly higher than that of the initial bacteria and recombinant bacteria 2. Recombinant bacteria 8 was named Yarrowia lipolytica XJ-CAP strain. In the production process of β-caryophyllene, since it is a fat-soluble product, a large amount of accumulation in cells can cause cytotoxicity. The traditional method is to add n-dodecane to the fermentation broth for biphasic fermentation, thereby simultaneously extracting toxic β-caryophyllene during the fermentation process to reduce the pressure on cells. However, n-dodecane itself has certain toxicity and can also have a negative impact on cell growth. To address this issue, this study reversed the natural promoter of the lipase gene TGL4 responsible for triglyceride degradation by knocking out the promoter of the gene. TGL4 Replaced with the native promoter P that is upregulated during the lipid accumulation phase YAT1 , realizing a new regulatory strategy. This strategy allows the accumulation of oil in the early growth stage of Yarrowia lipolytica for the storage of β-caryophyllene; in the late oil production stage, when lipids accumulate in large quantities, P YAT1 The promoter activated the expression of TGL4, and part of the triglycerides were degraded. Subsequently, through the activation of endogenous fatty acids and the β-oxidation pathway, a large amount of acetyl-CoA was generated for the synthesis of β-caryophyllene. The fermentation results showed that this strategy increased the yield of β-caryophyllene by nearly 2 times.

[0102] Table 3 β-caryophyllene production of initial bacteria, recombinant bacteria 2, recombinant bacteria 7, and recombinant bacteria 8

[0103] Fermentation method strain Biomass (g / L) β-Caryophyllene yield (g / L) Shake bottle Initial bacteria 4.02 0 Shake bottle Recombinant bacteria 2 5.03 0.26 Shake bottle Recombinant bacteria 7 4.81 0.82 Shake bottle Recombinant bacteria 8 5.22 1.52 Fermentation tank Recombinant bacteria 8 92.42 6.27

[0104] The recombinant bacteria 8 was inoculated into 50 mL of seed culture solution (YPD medium) and cultured at 30°C and 220 rpm for 24 hours to obtain seed solution, and then the seed solution was inoculated into a 5L fermenter at an inoculation amount of 5% v / v. The 5L fermenter was filled with 2.5L of fermentation medium, and the fermentation was carried out for 0-48 hours, and the dissolved oxygen was controlled at 20%; when the fermentation time was greater than 48 hours, the dissolved oxygen was controlled at 0-5%. During the fermentation process, the pH value was constantly controlled at 5.5 until the fermentation was completed. Feed was added once at 40 hours and 96 hours of fermentation time, and 800 mL of an aqueous solution containing 11.1 g of ammonium sulfate and 900 g of glucose per liter was added each time. The fermentation temperature was 28°C and the culture time was 6 days. Among them, the components of the fermentation medium are glucose 150g / L, ammonium sulfate 30g / L, yeast extract 3g / L, corn peptone 0.1g / L, potassium dihydrogen phosphate 4g / L, magnesium sulfate 2g / L, calcium sulfate 0.8g / L, sodium chloride 0.4g / L, ammonium sulfate hydrochloride 12mg / L, biotin 1mg / L, sodium molybdate 160mg / L, copper sulfate 0.2mg / L, boric acid 40mg / L, manganese sulfate 180mg / L and ferrous chloride 75mg / L. After 5 days of fermentation, the β-caryophyllene production of recombinant bacteria 8 reached a maximum of 6.27g / L, that is, 6.27g of β-caryophyllene per liter of fermentation liquid (Table 3), which is significantly higher than the initial bacteria and recombinant bacteria 2.

[0105] The present invention provides a recombinant Yarrowia lipolytica with high yield of β-caryophyllene and a construction method and application thereof. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A recombinant Yarrowia lipolytica with high yield of β-caryophyllene, characterized in that: The recombinant Yarrowia lipolytica expresses a heterologous gene; the heterologous gene is a gene encoding β-caryophyllene synthase or its E353D mutant from Artemisia annua; Among them, the nucleotide sequence of the β-caryophyllene synthase encoding gene derived from Artemisia annua is shown as SEQ ID No.1, and the nucleotide sequence of the β-caryophyllene synthase E353D mutant encoding gene derived from Artemisia annua is shown as SEQ ID No.

2.

2. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The recombinant Yarrowia lipolytica expresses 1-3 copies of heterologous genes.

3. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The recombinant Yarrowia lipolytica simultaneously expresses any one or more combinations of the 3-hydroxy-3-methylglutaryl CoA reductase encoding gene tHMG1, the isopentenyl pyrophosphate isomerase encoding gene IDI and the farnesyl pyrophosphate synthase encoding gene ERG20.

4. The recombinant Yarrowia lipolytica according to claim 3, characterized in that The GenBank accession number of the 3-hydroxy-3-methylglutaryl CoA reductase encoding gene tHMG1 is XP_503558.1, the GenBank accession number of the isopentenyl pyrophosphate isomerase encoding gene IDI is YALI0F04015g, and the GenBank accession number of the farnesyl pyrophosphate synthase encoding gene ERG20 is YALI0E05753g.

5. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The recombinant Yarrowia lipolytica simultaneously expresses the endogenous promoter P of the lipase encoding gene TGL4 TGL4 Replace with P YAT1 ; The P YAT1 The nucleotide sequence is shown in SEQ ID No.

3.

6. A recombinant Yarrowia lipolytica strain with high β-caryophyllene production, characterized in that: The recombinant Yarrowia lipolytica expresses two copies of the gene encoding the E353D mutant of β-caryophyllene synthase from Artemisia annua, and simultaneously expresses the 3-hydroxy-3-methylglutaryl CoA reductase encoding gene tHMG1, the isopentenyl pyrophosphate isomerase encoding gene IDI and the farnesyl pyrophosphate synthase encoding gene ERG20, and the endogenous promoter P of the lipase encoding gene TGL4 is expressed in the recombinant Yarrowia lipolytica. TGL4 Replace with P YAT1 ; The nucleotide sequence of the gene encoding the β-caryophyllene synthase E353D mutant from Artemisia annua is shown in SEQ ID No. 2, the GenBank accession number of the 3-hydroxy-3-methylglutaryl CoA reductase encoding gene tHMG1 is XP_503558.1, the GenBank accession number of the isopentenyl pyrophosphate isomerase encoding gene IDI is YALI0F04015g, the GenBank accession number of the farnesyl pyrophosphate synthase encoding gene ERG20 is YALI0E05753g, and the P YAT1 The nucleotide sequence is shown in SEQ ID No.

3.

7. The method for constructing a recombinant Yarrowia lipolytica according to any one of claims 1 to 6, characterized in that: The coding gene of the beta-caryophyllene synthase or its E353D mutant is integrated into the yeast genome in the form of a gene expression box.

8. The construction method according to claim 7, characterized in that: The steps include: (1) Construction of a heterologous gene expression cassette: amplifying the promoter, heterologous gene and terminator to obtain a promoter gene fragment, a heterologous gene fragment and a terminator gene fragment, respectively, and simultaneously ligating them into a plasmid vector to obtain a recombinant plasmid vector containing the heterologous gene expression cassette; (2) Introducing the recombinant plasmid vector containing the heterologous gene expression cassette into Yarrowia lipolytica, so that the gene expression cassette is integrated into the Yarrowia lipolytica genome.

9. The construction method according to claim 8, characterized in that: The construction method comprises step (3), wherein step (3) is to construct any one or more of a tHMG1 expression cassette, an IDI expression cassette, an ERG20 expression cassette and a promoter replacement expression cassette, and integrate the cassettes into the genome of Yarrowia lipolytica constructed in step (2).

10. The construction method according to claim 9, characterized in that: In step (3), the promoter replacement expression cassette comprises the upstream homology arm of the promoter of the lipase encoding gene TGL4, the promoter P YAT1 and the homology arm downstream of the start codon of the lipase encoding gene TGL4.

11. Use of the recombinant Yarrowia lipolytica according to any one of claims 1 to 6 in fermentation production of β-caryophyllene.

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