CN117327725A - Yarrowia lipolytica genetically engineered bacterium for producing beta-elemene, construction method and preparation method of beta-elemene - Google Patents

Yarrowia lipolytica genetically engineered bacterium for producing beta-elemene, construction method and preparation method of beta-elemene Download PDF

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CN117327725A
CN117327725A CN202310774091.3A CN202310774091A CN117327725A CN 117327725 A CN117327725 A CN 117327725A CN 202310774091 A CN202310774091 A CN 202310774091A CN 117327725 A CN117327725 A CN 117327725A
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plasmid
gene
yarrowia lipolytica
hbsbt01
elemene
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刘顺成
袁丽杰
徐龙兴
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North China University of Science and Technology
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Priority to PCT/CN2023/103738 priority patent/WO2023174453A2/en
Priority to ZA2024/00095A priority patent/ZA202400095B/en
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Abstract

The invention relates to a yarrowia lipolytica genetically engineered bacterium for producing beta-elemene, a construction method and a preparation method of the beta-elemene. Construction method the yarrowia lipolytica endogenous HMGR gene, optimized germacreneA synthase HaGAS L101WT412SD433NW436A Gene, ERG19 gene endogenous to yarrowia lipolytica, ERG13 endogenous to yarrowia lipolytica, haGAS of farnesyl diphosphate synthase ERG20 L101WT412SD433NW436A And integrating the LERG20 fusion gene into yarrowia lipolytica to obtain the genetically engineered yarrowia lipolytica strain for producing beta-elemene. The method can enable the carbon flux to flow into the synthesis way of the beta-elemene as much as possible, reduce the waste of the substrate and greatly improve the yield of the beta-elemene; the engineering bacteria have good subculture stability and can be used for large-scale commercial production.

Description

Yarrowia lipolytica genetically engineered bacterium for producing beta-elemene, construction method and preparation method of beta-elemene
Technical Field
The invention relates to the technical field of genetic engineering, in particular to yarrowia lipolytica genetic engineering bacteria for producing beta-elemene, a construction method and a preparation method of the beta-elemene.
Background
Beta-elemene (beta-elemene) (1S, 2S, 4R) - (-) -2, 4-diisopropenyl-1-methyl-1-vinylcyclohexane) is a sesquiterpenoid which is present in plants of the Compositae family such as dandelion. High purity beta-elemene is a transparent liquid with volatile odor, which is soluble in organic solvents such as alkanes and is practically insoluble in water. Beta-elemene has biological activities such as anti-inflammation, antioxidation, anticancer and the like, and has been applied to clinical treatment of cancers at present.
At present, the beta-elemene is mainly obtained by chemical synthesis, plant extraction or microbial fermentation. The chemical synthesis means has the advantages of complex technical route, high cost and low yield. The beta-elemene with biological activity can be obtained by extracting from plants, but the extraction rate is low, meanwhile, the growth period of the plants is long, the plant growth environment is harsh, and the factors limit the acquisition of the beta-elemene.
The method provides another mode for the production of the beta-elemene by metabolic engineering microorganisms, and has the advantages of low raw material cost, small environmental pollution and the like. Bacteria or yeast are transformed by metabolic engineering means, a heterologous biosynthesis way is introduced, and metabolic network optimization is combined to produce the beta-elemene with safety and high bioactivity.
Yarrowia lipolytica is a safe strain with wide substrate utilization as a oleaginous yeast. However, to date, few reports on the production of beta-elemene by yeast are available, the yield is generally low, and no research on the synthesis of beta-elemene in yarrowia lipolytica is available. Therefore, there is a need to develop engineering strains of yarrowia lipolytica for the synthesis of beta-elemene.
Disclosure of Invention
The invention aims to solve the technical problem of providing a yarrowia lipolytica genetically engineered bacterium for producing beta-elemene, a construction method and a preparation method of the beta-elemene.
The technical scheme for solving the technical problems is as follows:
the invention provides a construction method of genetically engineered yarrowia lipolytica for producing beta-elemene, which comprises the steps of carrying out endogenous HMGR gene of yarrowia lipolytica and optimizing HaGAS of germacrene A synthetase L101WT412SD433NW436A Gene, ERG19 gene endogenous to yarrowia lipolytica, ERG13 endogenous to yarrowia lipolytica, haGAS of farnesyl diphosphate synthase ERG20 L101WT412SD433NW436A The LERG20 fusion gene is integrated into yarrowia lipolytica to obtain the genetically engineered yarrowia lipolytica strain producing beta-elemene;
wherein the HaGAS L101WT412SD433NW436A The fusion gene of LERG20 is shown as SEQ ID No.1, and the optimized germacrene A synthetase HaGAS L101WT412SD433NW436A The sequence of the gene fragment is shown in SEQ ID No. 7.
Further, the method comprises the following steps:
s1, firstly introducing the HMGR gene into yarrowia lipolytica to obtain engineering bacteria HBSBT01-01; and then the optimized germacrene A synthetase HaGAS is carried out L101WT412SD433NW436A Introducing the gene fragment into the engineering bacterium HBSBT01-01 to obtain engineering bacterium HBSBT01-02;
s2, the ERG13 gene, the ERG19 gene and the HaGAS are combined L101WT412SD433NW436A Integrating the LERG20 fusion gene into the engineering bacterium HBSBT01-02 to obtain engineering bacterium HBSBT01-03;
s3, converting the plasmid pUB4-CRE into the engineering bacterium HBSBT01-12 to obtain engineering bacterium HBSBT01-13;
s4, integrating CAT2 genes endogenous to yarrowia lipolytica into the engineering bacterium HBSBT01-03 to obtain engineering bacterium HBSBT01-04;
s5, integrating an ERG19 gene, an HMGR gene and an ERG13 gene containing a peroxisome positioning tag ePTS1 into the engineering bacterium HBSBT01-04 to obtain engineering bacterium HBSBT01-05;
s6, ERG8 gene, ERG12 gene, IDI and HaGAS containing peroxisome location label ePTS1 L101WT412SD433NW436A And integrating the LERG20 gene into the engineering bacterium HBSBT01-05 to obtain engineering bacterium SBT01-06, wherein the engineering bacterium 06 is the yarrowia lipolytica genetic engineering bacterium for producing the beta-elemene.
Further, the step S1 specifically includes the following steps:
s1-1, connecting the HMGR gene fragment to a plasmid pHR_A08_hrGFP to obtain a plasmid pHR_A08_HMGR;
s1-2, converting the plasmid pHR_A08_HMGR and the plasmid pCRISPRyl_A08 into yarrowia lipolytica to obtain the engineering bacterium HBSBT01-01;
s1-3, the optimized germacrene A synthase HaGAS L101WT412SD433NW436A The gene fragment was ligated into the plasmid pHR_A08_hrGFP to give plasmid pHR_XPR2_HaGAS L101WT412SD433NW436A ；
S1-4, plasmid pHR_XPR2_HaGAS L101WT412SD433NW436A And converting the plasmid pCRISPRyl_A08 into the engineering bacterium HBSBT01-01 to obtain the engineering bacterium HBSBT01-02.
Further, the step S2 specifically includes the following steps:
s2-1, constructing an expression cassette P by adopting the ERG13 gene, a promoter PUB8T and a terminator CYC1T UB8T -sequence fragment of ERG13-CYC1t, and then the expression cassette P UB8T The sequence fragment of ERG13-CYC1t is ligated into plasmid pUC19, yielding plasmid pUC19-P UB8T -ERG13-CYC1t；
S2-2, constructing expression cassette P by adopting ERG19 gene, promoter PGPD and terminator XPR2t GPD -sequence fragment of ERG19-XPR2t, and then the expression cassette P GPD The sequence fragment of ERG19-XPR2t was ligated into plasmid pUC19 to give plasmid pUC19-P GPD -ERG19-XPR2t；
S2-3, use of the yarrowia lipolytica endogenous promoter P FBA The HaGAS L101WT412SD433NW436A Construction of expression cassette P by LERG20 fusion gene and terminator PEX20t FBA -HaGAS L101WT412SD433NW436A Sequence fragments of LERG20-PEX20t, and then the expression cassette P FBA -HaGAS L101WT412SD433NW436A The sequence fragment of LERG20-PEX20t was ligated into plasmid pUC19 to obtain plasmid pUC19-P FBA -HaGAS L101WT412SD433NW436A LERG20-PEX20t；
S2-4, using plasmid pINA1312 as a template, and amplifying to obtain LOXP-URA3d1-LOXP; seamlessly connecting a homologous arm at the upstream and downstream of a yarrowia lipolytica rDNA locus and a fragment LOXP-URA3d1-LOXP into a plasmid pUC19 to obtain a plasmid pUC19-rDNA-LUL;
s2-5, after the sequencing verification is correct, the expression cassette P is used for UB8T -ERG13-CYC1t, said expression cassette P GPD -ERG19-XPR2t and said expression cassette P FBA -HaGAS L101WT412SD433NW436A After the LERG20-PEX20t is amplified and purified from a corresponding plasmid, the plasmid pUC19-rDNA-LUL is seamlessly connected to the plasmid pUC19-rDNA-LUL, and a plasmid pUC19-rDNA-LUL-U13C-G19X-HG LEP with a sequence shown as SEQ ID No.2 is obtained;
s2-6, converting the plasmid pUC19-rDNALUL-U13C-G19X-HG LEP into the engineering bacterium HBSBT01-02 to obtain the engineering bacterium HBSBT01-03.
Further, the step S4 specifically includes the following steps:
s4-1, connecting a gene CAT2 fragment derived from yarrowia lipolytica into a plasmid pINA1312 to obtain a plasmid pINA1312-CAT2 with a sequence shown in SEQ ID No. 3;
s4-2, converting the plasmid pINA1312-CAT2 into engineering bacteria HBSBT01-03, and constructing to obtain the engineering bacteria HBSBT01-04.
Further, the sequences of the peroxisome localization tags ePTS1 in the step S5 and the step S6 are shown in SEQ ID No. 4.
Further, the step S5 specifically includes the following steps:
s5-1, respectively adding genes of peroxisome localization signals ePTS1 to 3' -ends of MVA pathway genes ERG19, HMGR and ERG13, and respectively constructing expression cassettes P GPD -ERG19-ePTS1-XPR2t, expression cassette P EXP -HMGR-ePTS1-MIGt, expression cassette P FBA -ERG13-ePTS1-PEX20t；
The expression cassette P GPD -ERG19-ePTS1-XPR2t, said expression cassette P EXP -HMGR-ePTS1-MIGt, said expression cassette P FBA -ERG13-ePTS1-PEX20t was seamlessly ligated into plasmid JMP114 to give plasmid JM618; the sequence of the plasmid JM618 is shown as SEQ ID No. 5;
s5-2, transforming the JM618 plasmid into a strain HBSBT01-04 to obtain engineering bacteria HBSBT01-05.
Further, the step S6 specifically includes the following steps:
s6-1, adding peroxisome localization signal ePTS1 to MVA pathway gene IDI and HaGAS respectively L101WT412SD433NW436A The 3' ends of LERG20, ERG8 and ERG12 are respectively constructed into expression cassettes P GPD -ERG8-ePTS1-XPR2t、P UB8T -ERG12-ePTS1-CYC1t, expression cassette P UB8T -IDI-ePTS1-CYC1t, expression cassette P EXP -HaGAS L101WT412SD433NW436A LERG20-ePTS1-MIGt；
The expression cassette P GPD -ERG8-ePTS1-XPR2t, said expression cassette P UB8T -ERG12-ePTS1-CYC1t, said expression cassette P UB8T -IDI-ePTS1-CYC1t, said expression cassette P EXP -HaGAS L101WT412SD433NW436A The LERG20-ePTS1-MIGt is seamlessly connected into a plasmid JM617 to obtain a plasmid JM620; the sequence of the plasmid JM620 is shown as SEQ ID No. 6.
S6-2, converting the plasmid JM620 into the engineering bacterium HBSBT01-16 to obtain the engineering bacterium SBT01-06.
The invention also provides a genetically engineered yarrowia lipolytica strain for producing the beta-elemene, which is obtained by adopting the construction method.
The invention also provides a preparation method of the beta-elemene, which adopts the genetically engineered yarrowia lipolytica bacteria for producing the beta-elemene.
The beneficial effects of the invention are as follows:
(1) The construction method of the yarrowia lipolytica genetically engineered bacterium for producing beta-elemene comprises the steps of constructing a germacrene A synthetase gene HaGAS L101WT412SD433NW436A Yarrowia lipolytica (Yarrowia lipolytica) endogenous 3-hydroxyThe gene HMGR of the acyl-3-methylglutaryl-CoA reductase is introduced into yarrowia lipolytica, and mevalonate pyrophosphate decarboxylase (ERG 19), S-3-hydroxy-3-methylglutaryl-CoA synthetase (ERG 13) and fusion expressed germacrene A synthetase (HaGAS) are integrated L101WT412SD433NW436A ) And HaGAS of farnesyl diphosphate synthase (ERG 20) L101WT412SD433NW436A LERG20; the carbon flux flows into the synthesis way of the beta-elemene as much as possible, reduces the waste of the substrate and greatly improves the yield of the beta-elemene;
(2) The construction method of the genetically engineered yarrowia lipolytica for producing the beta-elemene further comprises the step of fusing the carnitine acetyl transferase CAT2 endogenous to the yarrowia lipolytica, so that the transformation of an acetyl coenzyme A synthesis path is realized;
(3) The invention relates to a construction method of a genetically engineered yarrowia lipolytica strain producing beta-elemene, which also comprises the steps of fusing S-3-hydroxy-3-methylglutaryl coenzyme A synthetase ERG13, mevalonate kinase ERG8, mevalonate pyrophosphoric acid decarboxylase ERG19 carrying peroxisome positioning label ePTS1, reducing HMG-CoA into endogenous HMGR of mevalonate, isopentenyl diphosphate delta-isomerase IDI and fusing expressed germacrene A synthetase (HaGAS L101WT412SD433NW436A ) And HaGAS of farnesyl diphosphate synthase (ERG 20) L101WT412SD433NW436A A step of LERG20; the biosynthesis path of the beta-elemene can be positioned in a peroxisome, and more production places are provided for the production of the beta-elemene, so that the yield of the beta-elemene can be greatly improved;
(4) The yarrowia lipolytica genetically engineered bacterium for producing the beta-elemene has high yield of the beta-elemene and good subculture stability of the strain, and can be used for large-scale commercial production;
(5) The preparation method of the beta-elemene adopts the running engineering bacteria to prepare and produce the beta-elemene, so that the beta-elemene has high yield, low cost, good purity and safe utilization, and has good industrialized application prospect.
Drawings
FIG. 1 is a schematic diagram showing the steps of metabolism of genetically engineered bacteria of yarrowia lipolytica producing beta-elemene of the present invention;
FIG. 2 is a graph showing comparison of stability of a strain in examples after continuous subculture in a construction method of a genetically engineered yarrowia lipolytica strain producing beta-elemene of the present invention.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
The invention relates to a construction method of genetically engineered yarrowia lipolytica for producing beta-elemene, which comprises the steps of carrying out endogenous HMGR gene (accession number is GB: NC_006071 in NCBI) of yarrowia lipolytica and optimized germacrene A synthetase HaGAS L101WT412SD433NW436A Gene, ERG19 gene endogenous to yarrowia lipolytica, ERG13 endogenous to yarrowia lipolytica, haGAS of farnesyl diphosphate synthase ERG20 L101WT412SD433NW436A And integrating the LERG20 fusion gene into yarrowia lipolytica to obtain the genetically engineered yarrowia lipolytica strain for producing beta-elemene.
Wherein HaGAS L101WT412SD433NW436A The sequence of the LERG20 fusion gene is shown as SEQ ID No.1, and the optimized germacrene A synthetase HaGAS L101WT412SD433NW436A The sequence of the gene fragment is shown in SEQ ID No. 7; in NCBI, the HMGR gene is numbered YALI0E04807g, the ERG19 gene is numbered YALI0F05632g, and the ERG13 gene is numbered YALI0F 30841 g.
The engineering strain constructed by the method contains 1 copy of the optimized sunflower (Helianthus annuus) source germacrene A synthetase gene HaGAS containing point mutation L101WT412SD433NW436A And one copy of the 3-hydroxy-3-methylglutaryl-CoA reductase gene HMGR endogenous to yarrowia lipolytica (Yarrowia lipolytica). The recombinant strain integrates mevalonate pyrophosphate decarboxylase (ERG 19), S-3-hydroxy-3-methylglutaryl-CoA synthetase (ERG 13) and fusion expressed germacrene A synthetase (HaGAS) L101WT412SD433NW436A ) And HaGAS of farnesyl diphosphate synthase (ERG 20) L101WT412SD433NW436A LERG20; make carbon lead toThe amount of the catalyst flows into the synthesis way of the beta-elemene as much as possible, reduces the waste of the substrate and can greatly improve the yield of the beta-elemene.
Preferably, the method of the invention further comprises the step of fusing the carnitine acetyl transferase CAT2 endogenous to yarrowia lipolytica, thereby realizing the modification of the synthesis path of acetyl-CoA.
Preferably, the method of the present invention further comprises fusing S-3-hydroxy-3-methylglutaryl coenzyme A synthetase ERG13 carrying peroxisome localization tag ePTS1, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, endogenous HMGR for reducing HMG-CoA to mevalonate, isopentenyl diphosphate delta-isomerase IDI and fusion expressing germacrene A synthetase (HaGAS) L101WT412SD433NW436A ) And HaGAS of farnesyl diphosphate synthase (ERG 20) L101WT412SD433NW436A Step of LERG 20. The obtained yarrowia lipolytica genetically engineered bacteria producing beta-elemene overexpresses the rate limiting step in the synthetic pathway (HMGR, ERG13, ERG19 and ERG20 in the MVA pathway and HaGAS) L101WT412SD433NW436A LERG 20). Therefore, the biosynthesis path of the beta-elemene can be positioned in the peroxisome, and more production sites are provided for the production of the beta-elemene, so that the yield of the beta-elemene can be greatly improved.
Wherein, in NCBI, the IDI gene is numbered as YALI0F04015g, the ERG20 gene is numbered as YALI0E05753g, the ERG8 gene is numbered as YALI0E06193g, and the ERG12 gene is numbered as YALI0B16038g.
Preferably, the starting strain of the invention is a wild-type yarrowia lipolytica which has not undergone any gene knockout.
FIG. 1 is a schematic diagram showing the metabolic steps of the engineering bacteria of the present invention.
The sequences of the primers used in the construction method of the yarrowia lipolytica genetic engineering bacteria producing the beta-elemene are shown in the table 1, and the specific steps of the method are as follows:
s1, firstly, introducing an HMGR gene into yarrowia lipolytica to obtain engineering bacteria HBSBT01-01; and then optimizing the germacrene A synthetase HaGAS L101WT412SD433NW436A Gene fragment introductionEngineering bacteria HBSBT01-01 to obtain engineering bacteria HBSBT01-02.
Preferably, the process of step S1 is specifically as follows:
s1-1, connecting the HMGR gene fragment to a plasmid pHR_A08_hrGFP through double digestion of NheI and BssHII to obtain a plasmid pHR_A08_HMGR, wherein the used primer is H-F/H-R.
S1-2, converting the plasmid pHR_A08_HMGR and the plasmid pCRISPRyl_A08 into yarrowia lipolytica to obtain engineering bacteria HBSBT01-01. Wherein, the transformation uses a kit Frozen EZ Yeast Transformation II TM (available from Zymo Research) was performed according to the method described in the kit.
S1-3, the optimized germacrene A synthase HaGAS L101WT412SD433NW436A The gene fragment was ligated into the plasmid pHR_A08_hrGFP by double cleavage with NheI and BssHII to give plasmid pHR_XPR2_HaGAS L101WT412SD433NW436A The primer used was HAGAS-F/HAGAS-R.
S1-4 plasmid pHR_XPR2_HaGAS L101WT412SD433NW436A And converting the plasmid pCRISPRyl_A08 into engineering bacteria HBSBT01-01 to obtain engineering bacteria HBSBT01-02. Wherein, the transformation uses a kit Frozen EZ Yeast Transformation II TM (available from Zymo Research) was performed according to the method described in the kit.
The above-mentioned plasmids pHR_A08_hrGFP, plasmid pCRISPRyl_A08, plasmid pHR_A08_hrGFP, plasmid pCRISPRyl_A08 are described in Schwartz, C.shabber-Hussain, M.f.true, K.Blnner, M.f.Wheeldon, I.2017.Standard markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth Biol,6 (3), 402-409.
S2, ERG13 gene, ERG19 gene and HaGAS L101WT412SD433NW436A The LERG20 fusion gene is integrated into engineering bacteria HBSBT01-02 to obtain engineering bacteria HBSBT01-03.
Preferably, the process of step S2 is specifically as follows:
s2-1, construction of expression cassette P by overlapping PCR with ERG13 Gene, promoter PUB8T and terminator CYC1T UB8T Sequence fragment of ERG13-CYC1t, and expression cassette P UB8T -ERG13-CYC1tThe sequence fragment was ligated into plasmid pUC19 by double digestion with EcoRI and HindIII to give plasmid pUC19-P UB8T ERG13-CYC1t, the primers used are U-F/R,13-F/R and C-F/R, respectively.
S2-2, construction of expression cassette P by overlapping PCR with ERG19 gene, promoter PGPD and terminator XPR2t GPD Sequence fragment of ERG19-XPR2t, expression cassette P GPD The sequence fragment of ERG19-XPR2t was ligated into plasmid pUC19 by double digestion with EcoRI and HindIII to give plasmid pUC19-P GPD ERG19-XPR2t, primers used were G-F/R,19-F/R and X-F/R, respectively.
S2-3, use of the yarrowia lipolytica endogenous promoter P FBA 、HaGAS L101WT412SD433NW436A Construction of expression cassette P by overlapping PCR of LERG20 fusion Gene and terminator PEX20t FBA -HaGAS L101WT412SD433NW436A Sequence fragment of LERG20-PEX20t, and expression cassette P FBA -HaGAS L101WT412SD433NW436A The sequence fragment of LERG20-PEX20t was ligated into plasmid pUC19 by double digestion with EcoRI and HindIII to give plasmid pUC19-P FBA -HaGAS L101WT412SD433NW436A LERG20-PEX20t, the primers used were B-F/R, HA-F/R, L-F/R and P-F/R, respectively.
S2-4, using a plasmid pINA1312 as a template, and amplifying to obtain LOXP-URA3d1-LOXP, wherein the used primer is LUL-F/LUL-R; the homologous arm at the upstream and downstream of the rDNA site of yarrowia lipolytica and the fragment LOXP-URA3d1-LOXP were seamlessly ligated into plasmid pUC19 to obtain plasmid pUC19-rDNA-LUL using the primers R-F-F/R and R-R-F/R.
The plasmid pINA1312 described above is referred to Nicaud, j.m.; madzak, c.; van den Broek, p.; gysler, c.; duboc, p.; niederberger, p.; gaillidin, C. (2002) Protein expression and secretion in the Yeast Yarrowia lipolytica. FEMS Yeast Res.2,371-379. The method of manufacture.
S2-5, after the sequencing verification is correct, the expression cassette P is used for UB8T -ERG13-CYC1t, expression cassette P GPD -ERG19-XPR2t and expression cassette P FBA -HaGAS L101WT412SD433NW436A After the LERG20-PEX20t is amplified and purified from the corresponding plasmid, the amplified and purified plasmid is seamlessly connected to a plasmid pUC19-rDNA-LUL to obtain a plasmid p with a sequence shown as SEQ ID No.2UC19-rDNA-LUL-U13C-G19X-HG LEP, and the primers are 1-F/R,2-F/R and 3-F/R.
S2-6, converting a plasmid pUC19-rDNALUL-U13C-G19X-HG LEP into engineering bacteria HBSBT01-02 to obtain engineering bacteria HBSBT01-03.
S3, converting the plasmid pUB4-CRE into engineering bacteria HBSBT01-12 to obtain engineering bacteria HBSBT01-13. Wherein, the transformation uses a kit Frozen EZ Yeast Transformation II TM (available from Zymo Research) was performed according to the method described in the kit.
S4, integrating CAT2 genes endogenous to yarrowia lipolytica into engineering bacteria HBSBT01-03 to obtain engineering bacteria HBSBT01-04.
Preferably, the process of step S4 is specifically as follows:
s4-1, the gene CAT2 fragment from yarrowia lipolytica is connected to plasmid pINA1312 by BamHI and Pml I double enzyme digestion to obtain plasmid pINA1312-CAT2 with the sequence shown in SEQ ID No.3, and the primer is CAT-F/R.
S4-2, linearizing the plasmid pINA1312-CAT2 through NotI, and then converting the linearized plasmid into engineering bacteria HBSBT01-03 to construct engineering bacteria HBSBT01-04. Wherein, the transformation uses a kit Frozen EZ Yeast Transformation II TM (available from Zymo Research) was performed according to the method described in the kit.
S5, integrating an ERG19 gene, an HMGR gene and an ERG13 gene containing a peroxisome location tag ePTS1 into engineering bacteria HBSBT01-04 to obtain engineering bacteria HBSBT01-05; wherein the sequence of the peroxisome localization tag ePTS1 is shown as SEQ ID No. 4.
Preferably, the process of step S5 is specifically as follows:
s5-1, respectively adding genes of peroxisome localization signals ePTS1 to 3' -ends of ERG19 genes, HMGR genes and ERG13 genes in MVA pathway, and respectively constructing expression cassettes P through overlapping PCR amplification GPD -ERG19-ePTS1-XPR2t, expression cassette P EXP -HMGR-ePTS1-MIGt, expression cassette P FBA ERG13-ePTS1-PEX20t, G-19-P-X-F/R, E-H-P-M-F/R and F-13-P-P-F/R.
Expression cassette P GPD -ERG19-ePTS1-XPR2t and expression cassette P EXP -HMGR-ePTS1-MIGt, expression cassette P FBA -ERG13-ePTS1-PEX20t is seamlessly connected into a plasmid JMP114 tangential to Acc65I enzyme to obtain a plasmid JM618; the sequence of plasmid JM618 is shown in SEQ ID No.5, and the primers used are 4-F/R,5-F/R and 6-F/R.
S5-2, transforming JM618 plasmid linearized by SwaI into a strain HBSBT01-04 to obtain engineering bacteria HBSBT01-05. Wherein, the transformation uses a kit Frozen EZ Yeast Transformation II TM (available from Zymo Research) was performed according to the method described in the kit.
S6, ERG8 gene, ERG12 gene, IDI and HaGAS containing peroxisome location label ePTS1 L101WT412SD433NW436A The LERG20 gene is integrated into engineering bacteria HBSBT01-05 to obtain engineering bacteria SBT01-06, and engineering bacteria 06 are yarrowia lipolytica genetic engineering bacteria producing beta-elemene.
Preferably, the process of step S6 is specifically as follows:
s6-1, adding peroxisome localization signal ePTS1 to IDI gene and HaGAS in MVA path respectively L101WT412SD433NW436A The 3' end of LERG20 gene, ERG8 gene and ERG12 gene are amplified by overlapping PCR to construct expression cassette P GPD -ERG8-ePTS1-XPR2t、P UB8T -ERG12-ePTS1-CYC1t, expression cassette P UB8T -IDI-ePTS1-CYC1t, expression cassette P EXP -HaGAS L101WT412SD433NW436A LERG20-ePTS1-MIGt, the primers used are I-P-F/R, GL20-P-F/R, 8-P-F/R and 12-P-F/R;
expression cassette P GPD -ERG8-ePTS1-XPR2t, expression cassette P UB8T -ERG12-ePTS1-CYC1t, expression cassette P UB8T -IDI-ePTS1-CYC1t, expression cassette P EXP -HaGAS L101WT412SD433NW436A The LERG20-ePTS1-MIGt is seamlessly connected into a plasmid JM617 tangential by Acc65I enzyme to obtain a plasmid JM620; the sequence of plasmid JM620 is shown in SEQ ID No.6, and the primers used are 7-F/R,8-F/R,9-F/R and 10-F/R.
S6-2, converting the plasmid JM620 linearized by NotI into engineering bacteria HBSBT01-16 to obtain engineering bacteria SBT01-06. Wherein the transformation uses a kit Frozen EZ YeastTransformation II TM (available from Zymo Research) was performed according to the method described in the kit.
TABLE 1 sequences corresponding to the primers
In the above steps, plasmid pUB4-CRE of step S3, plasmid JMP114 of step S5, and plasmid JM617 of step 6 were prepared as described in Fickers, P., le Dall, M.T., gaillidine, C., thonart, P., and Nicaud, J.M. (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica.J.Microbiol. Methods 55,727-737.
In the above steps, each expression cassette, promoter and terminator were prepared as described in Celinska, E.Ledesma-Amaro, R.Larroude, M.Rossicol, T.Pauthenier, C.and Nicaud, J.M. (2017) Golden gate assembly system dedicated to complex pathway manipulation in Yarrowia lipotics. Microb.Biotechnol.10,450-455.
The yarrowia lipolytica genetically engineered bacterium for producing the beta-elemene is obtained by adopting the construction method.
The preparation method of the beta-elemene adopts the yarrowia lipolytica genetic engineering bacteria for producing the beta-elemene.
The invention is illustrated by the following specific examples:
examples
The method of the invention is adopted to construct the yarrowia lipolytica genetic engineering strain for producing the beta-elemene. The starting strain used in this example was commercially available wild-type MYA-2613.
The enzymes, kits, plasmids used in this example were:
Hi-Fi enzyme, taq enzyme, was purchased from Norpran under the product numbers Phanta Super-Fidel ity DNA Polymerase (P501) and 2 XTaq Master Mix (Dye Plus), respectively. Kit for homologous recombination was purchased from Northenzan under the accession numberII One Step Cloning Kit(C112)。
The yarrowia lipolytica beta-elemene which is obtained by the embodiment has high yield of beta-elemene, and can lead the beta-elemene to reach the yield of over 1350mg/L in the shake flask level.
The detection mode is as follows:
1ml of dodecane was added to 50ml of the fermentation broth at the beginning of fermentation, and the fermentation broth was collected by centrifugation for 72 hours to collect the upper dodecane layer. The dodecane layer obtained by centrifugation was mixed 1:1 with a dodecane solution of β -caryophyllene at 100 mg/L. The samples were filtered using an organic phase filter head and then detected using GC-MS (gas chromatography-mass spectrometry). The column was a DB-5HT GC column (30 mm. Times.0.25 mm,0.1 μm) with the following protocol: 140℃for 2 minutes, raised to 170℃at a rate of 5℃per minute, held for 1 minute, and then raised to 280℃at a rate of 20℃per minute.
Meanwhile, as can be seen from fig. 2, after passage of 20 generations, the strain provided by the invention has good stability in both yield and biomass, which means that the strain obtained by the embodiment has good stability after continuous passage culture, can be used for large-scale commercial production, and the obtained beta-elemene can be safely utilized and has good industrial application prospect.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (10)

1. Yarrowia lipolytica genetic engineering for producing beta-elemeneA method for constructing a Protous comprising the steps of providing a yarrowia lipolytica endogenous HMGR gene and an optimized germacrene A synthase HaGAS L101WT412SD433NW436A Gene, ERG19 gene endogenous to yarrowia lipolytica, ERG13 endogenous to yarrowia lipolytica, haGAS of farnesyl diphosphate synthase ERG20 L101WT412SD433NW436A The LERG20 fusion gene is integrated into yarrowia lipolytica to obtain the genetically engineered yarrowia lipolytica strain producing beta-elemene;
wherein the HaGAS L101WT412SD433NW436A The fusion gene of LERG20 is shown as SEQ ID No.1, and the optimized germacrene A synthetase HaGAS L101WT412SD433NW436A The sequence of the gene fragment is shown in SEQ ID No. 7.
2. The method for constructing genetically engineered yarrowia lipolytica bacteria producing beta-elemene as claimed in claim 1, which is characterized by comprising the following steps:
s1, firstly introducing the HMGR gene into yarrowia lipolytica to obtain engineering bacteria HBSBT01-01; and then the optimized germacrene A synthetase HaGAS is carried out L101WT412SD433NW436A Introducing the gene fragment into the engineering bacterium HBSBT01-01 to obtain engineering bacterium HBSBT01-02;
s2, the ERG13 gene, the ERG19 gene and the HaGAS are combined L101WT412SD433NW436A Integrating the LERG20 fusion gene into the engineering bacterium HBSBT01-02 to obtain engineering bacterium HBSBT01-03;
s3, converting the plasmid pUB4-CRE into the engineering bacterium HBSBT01-12 to obtain engineering bacterium HBSBT01-13;
s4, integrating CAT2 genes endogenous to yarrowia lipolytica into the engineering bacterium HBSBT01-03 to obtain engineering bacterium HBSBT01-04;
s5, integrating an ERG19 gene, an HMGR gene and an ERG13 gene containing a peroxisome positioning tag ePTS1 into the engineering bacterium HBSBT01-04 to obtain engineering bacterium HBSBT01-05;
s6, ERG8 gene, ERG12 gene, IDI and HaGAS containing peroxisome location label ePTS1 L101WT412SD433NW436A The LERG20 gene is integrated into the engineering bacterium HBSBT01-05 to obtainEngineering bacteria SBT01-06, wherein the engineering bacteria 06 are genetically engineered bacteria of yarrowia lipolytica for producing beta-elemene.
3. The method for constructing genetically engineered yarrowia lipolytica bacteria producing beta-elemene according to claim 2, wherein the step S1 is specifically as follows:
s1-1, connecting the HMGR gene fragment to a plasmid pHR_A08_hrGFP to obtain a plasmid pHR_A08_HMGR;
s1-2, converting the plasmid pHR_A08_HMGR and the plasmid pCRISPRyl_A08 into yarrowia lipolytica to obtain the engineering bacterium HBSBT01-01;
s1-3, the optimized germacrene A synthase HaGAS L101WT412SD433NW436A The gene fragment was ligated into the plasmid pHR_A08_hrGFP to give plasmid pHR_XPR2_HaGAS L101WT412SD433NW436A ；
S1-4, plasmid pHR_XPR2_HaGAS L101WT412SD433NW436A And converting the plasmid pCRISPRyl_A08 into the engineering bacterium HBSBT01-01 to obtain the engineering bacterium HBSBT01-02.
4. The method for constructing genetically engineered yarrowia lipolytica bacteria producing beta-elemene according to claim 3, wherein the step S2 is specifically as follows:
s2-1, using the ERG13 gene and promoter P UB8T And terminator CYC1t to construct expression cassette P UB8T -sequence fragment of ERG13-CYC1t, and then the expression cassette P UB8T The sequence fragment of ERG13-CYC1t is ligated into plasmid pUC19, yielding plasmid pUC19-P UB8T -ERG13-CYC1t；
S2-2, using the ERG19 gene and promoter P GPD And terminator XPR2t to construct expression cassette P GPD -sequence fragment of ERG19-XPR2t, and then the expression cassette P GPD The sequence fragment of ERG19-XPR2t was ligated into plasmid pUC19 to give plasmid pUC19-P GPD -ERG19-XPR2t；
S2-3, use of the yarrowia lipolytica endogenous promoter P FBA The HaGAS L101WT412SD433NW436A LERG20 fusion geneConstruction of expression cassette P by terminator PEX20t FBA -HaGAS L101WT412SD433NW436A Sequence fragments of LERG20-PEX20t, and then the expression cassette P FBA -HaGAS L101WT412SD433NW436A The sequence fragment of LERG20-PEX20t was ligated into plasmid pUC19 to obtain plasmid pUC19-P FBA -HaGAS L101WT412SD433NW436A LERG20-PEX20t；
S2-4, using plasmid pINA1312 as a template, and amplifying to obtain LOXP-URA3d1-LOXP; seamlessly connecting a homologous arm at the upstream and downstream of a yarrowia lipolytica rDNA locus and a fragment LOXP-URA3d1-LOXP into a plasmid pUC19 to obtain a plasmid pUC19-rDNA-LUL;
s2-5, after the sequencing verification is correct, the expression cassette P is used for UB8T -ERG13-CYC1t, said expression cassette P GPD -ERG19-XPR2t and said expression cassette P FBA -HaGAS L101WT412SD433NW436A After the LERG20-PEX20t is amplified and purified from a corresponding plasmid, the plasmid pUC19-rDNA-LUL is seamlessly connected to the plasmid pUC19-rDNA-LUL, and a plasmid pUC19-rDNA-LUL-U13C-G19X-HG LEP with a sequence shown as SEQ ID No.2 is obtained;
s2-6, converting the plasmid pUC19-rDNALUL-U13C-G19X-HG LEP into the engineering bacterium HBSBT01-02 to obtain the engineering bacterium HBSBT01-03.
5. The method for constructing genetically engineered yarrowia lipolytica bacteria producing beta-elemene according to claim 4, wherein the step S4 is specifically as follows:
s4-1, connecting a gene CAT2 fragment derived from yarrowia lipolytica into a plasmid pINA1312 to obtain a plasmid pINA1312-CAT2 with a sequence shown in SEQ ID No. 3;
s4-2, converting the plasmid pINA1312-CAT2 into engineering bacteria HBSBT01-03, and constructing to obtain the engineering bacteria HBSBT01-04.
6. The method for constructing genetically engineered yarrowia lipolytica bacteria producing beta-elemene according to claim 5, wherein the sequences of peroxisome location tags ePTS1 in the step S5 and the step S6 are shown in SEQ ID No. 4.
7. The method for constructing genetically engineered yarrowia lipolytica bacteria producing beta-elemene according to claim 6, wherein the step S5 is specifically as follows:
s5-1, respectively adding genes of peroxisome localization signals ePTS1 to 3' ends of ERG19 genes, HMGR genes and ERG13 genes, and respectively constructing expression cassettes P GPD -ERG19-ePTS1-XPR2t, expression cassette P EXP -HMGR-ePTS1-MIGt, expression cassette P FBA -ERG13-ePTS1-PEX20t；
The expression cassette P GPD -ERG19-ePTS1-XPR2t, said expression cassette P EXP -HMGR-ePTS1-MIGt, said expression cassette P FBA -ERG13-ePTS1-PEX20t was seamlessly ligated into plasmid JMP114 to give plasmid JM618; the sequence of the plasmid JM618 is shown as SEQ ID No. 5;
s5-2, transforming the JM618 plasmid into a strain HBSBT01-04 to obtain engineering bacteria HBSBT01-05.
8. The method for constructing genetically engineered yarrowia lipolytica bacteria producing beta-elemene according to claim 7, wherein the step S6 is specifically as follows:
s6-1, adding peroxisome localization signal ePTS1 to IDI gene and HaGAS respectively L101WT412SD433NW436A The 3' end of the LERG20 gene, the ERG8 gene and the ERG12 gene are respectively constructed into an expression cassette P GPD -ERG8-ePTS1-XPR2t、P UB8T -ERG12-ePTS1-CYC1t, expression cassette P UB8T -IDI-ePTS1-CYC1t, expression cassette P EXP -HaGAS L101WT412SD433NW436A LERG20-ePTS1-MIGt；
The expression cassette P GPD -ERG8-ePTS1-XPR2t, said expression cassette P UB8T -ERG12-ePTS1-CYC1t, said expression cassette P UB8T -IDI-ePTS1-CYC1t, said expression cassette P EXP -HaGAS L101WT412SD433NW436A The LERG20-ePTS1-MIGt is seamlessly connected into a plasmid JM617 to obtain a plasmid JM620; the sequence of the plasmid JM620 is shown as SEQ ID No. 6;
s6-2, converting the plasmid JM620 into the engineering bacterium HBSBT01-16 to obtain the engineering bacterium SBT01-06.
9. The genetically engineered yarrowia lipolytica strain producing beta-elemene is characterized in that the genetically engineered yarrowia lipolytica strain producing beta-elemene is obtained by adopting the construction method of any one of claims 1-8.
10. The method for preparing the beta-elemene is characterized in that the beta-elemene-producing yarrowia lipolytica genetic engineering strain as claimed in claim 9 is adopted for preparation.
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