Yeast gene engineering strain for producing parthenolide and construction method thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a yeast gene engineering strain for producing parthenolide and a construction method of the yeast gene engineering strain. According to the present invention, it is determined that the improvement of the heme supply in the yeast gene engineering strain for producing parthenolide is beneficial to the improvement of the parthenolide yield, and it is proved that the integration site of the heme synthesis gene has effects on the parthenolide yield, such that the powerful evidence and the basis are provided for the subsequent continuous optimization and parthenolide yield increasing.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a genetically engineered yeast strain for producing parthenolide and a construction method thereof. Background Art

[0002] Parthenolide is a sesquiterpene lactone and a direct precursor of the anti-glioblastoma drug ACT001. Currently, the sources of parthenolide include extraction from plants and chemical synthesis. Since parthenolide has strict requirements on the growth environment, the area suitable for growth is limited, and its growth cycle is long, the plant extraction method limits the large-scale supply of raw materials. At present, the yield of parthenolide extracted from dried parthenolide leaves and magnolia root bark is low, and the yield is easily affected by factors such as climate change, pests and diseases, resulting in poor stability of raw material supply and difficulty in meeting large-scale production needs. The content of parthenolide in parthenolide is relatively low, and generally a large amount of raw materials is required to extract a certain amount of product, which increases the extraction cost and workload. It is technically difficult to efficiently and highly purifiedly separate parthenolide from complex plant components. The current extraction and separation methods such as solvent extraction and supercritical fluid extraction need to be further improved in purity and yield. In the chemical synthesis pathway, due to the complex chemical structure of parthenolide, it is challenging to develop an efficient synthesis route, which often requires multi-step reactions, harsh reaction conditions, and difficult to control the stability and yield of intermediates. Whether extracted from natural plants or chemically synthesized, the production process of parthenolide involves high costs, including raw material procurement, equipment investment, energy consumption, technology research and development, etc., resulting in expensive products and limiting its large-scale application. A large amount of organic solvents and chemical reagents may be used in the chemical synthesis process, and the wastewater, waste gas, waste residue, etc. generated will cause certain pollution to the environment, facing greater environmental pressure, and additional costs need to be invested for environmental protection treatment. Summary of the invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a genetically engineered yeast strain for producing parthenolide and a construction method thereof. The present invention provides a method for transforming a brewer's yeast strain using genetic engineering to further increase the yield of parthenolide.

[0004] The present invention provides the use of at least one of the following (1) to (4) in increasing the production of parthenolide in recombinant Saccharomyces cerevisiae:

[0005] (1) Gene encoding HEM2;

[0006] (2) Mutant PTS of parthenolide synthase PTS Y22G genes;

[0007] (3) the gene encoding HRD1;

[0008] (4) TKL2 locus as a gene integration site;

[0009] The recombinant Saccharomyces cerevisiae is SyBE_Sc07140053.

[0010] Among them, the Gene ID of HEM2 is 852842, PTS Y22G The gene encoding HRD1 has a nucleotide sequence as shown in SEQ ID NO:1, and the gene encoding HRD1 has a nucleotide sequence as shown in SEQ ID NO:2.

[0011] The present invention provides an expression cassette, comprising at least one of expression cassettes 1 to 4

[0012] The expression cassette 1 includes TEF1-PTS Y22G -HXT7t;

[0013] The expression cassette 2 includes TDH3p-HRD1-TDH1t;

[0014] The expression cassette 3 includes TEF1p-Y22G-HXT7t-TDH3p-HRD1-TDH1T;

[0015] The expression cassette 4 includes TEF1p-HEM2-HXT7t.

[0016] In some embodiments, the expression cassette 4 is expressed at the TKL2 site.

[0017] The invention provides an expression vector, comprising a backbone vector and the expression cassette.

[0018] The present invention provides a host cell, comprising the expression cassette and / or the expression vector;

[0019] The host cell includes Saccharomyces cerevisiae.

[0020] The present invention provides a recombinant strain, wherein the expression cassette and / or the expression vector are transformed into a chassis strain.

[0021] In some embodiments, the chassis strain is recombinant Saccharomyces cerevisiae SyBE_Sc07140053, and the gene encoding HEM2 is integrated into the TKL2 site of the chassis strain.

[0022] The present invention provides a method for constructing the recombinant strain, comprising genetically modifying the chassis strain, wherein the modification comprises the modification shown in ①, or further comprises the modification shown in ② and / or ③ on the basis of ①:

[0023] ①, transfer the coding gene of HEM2;

[0024] ②, overexpression of the mutant PTS of parthenolide synthase PTS Y22G genes;

[0025] ③, overexpression of the gene encoding HRD1;

[0026] Preferably, the gene encoding HEM2 is integrated into the TKL2 site of the chassis strain.

[0027] The present invention provides the use of at least one of the following I to V in the synthesis of parthenolide:

[0028] Ⅰ, the expression cassette;

[0029] II. the expression vector;

[0030] III. the host cell;

[0031] IV. the recombinant strain;

[0032] V. The recombinant strain constructed by the construction method.

[0033] The invention provides a method for synthesizing parthenolide, comprising culturing the recombinant strain or the recombinant strain constructed by the construction method to obtain parthenolide.

[0034] The present invention determines that increasing the heme supply in a genetically engineered yeast strain producing parthenolide is beneficial to increasing the yield of parthenolide, and at the same time proves that the integration site of the heme synthesis gene also has an impact on the yield of parthenolide, providing strong evidence and basis for subsequent optimization and improvement of parthenolide production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram showing the pathway of heme synthesis in yeast;

[0036] Figure 2 Schematic diagram showing the pathway for recombinant yeast to produce parthenolide;

[0037] Figure 3 A process diagram showing the construction of the heme enhancement module;

[0038] Figure 4 Schematic diagram showing the construction of the functional expression cassette WJ416k;

[0039] Figure 5 Schematic diagram showing the construction of plasmid pYTQ090;

[0040] Figure 6 Schematic diagram showing the construction of plasmid pYTQ092;

[0041] Figure 7 Schematic diagram showing the construction of plasmid pYTQ093;

[0042] Figure 8 Schematic diagram showing the construction of plasmid pCRI2;

[0043] Fig. 9 Schematic diagram showing the construction of plasmid pYTQ094;

[0044] Fig.10 Schematic diagram showing the construction of pZK028;

[0045] Fig.11 The figure shows the fermentation statistics of the production of parthenolide by 4 strains of Saccharomyces cerevisiae into which the plasmid overexpressing the heme gene was introduced;

[0046] Fig.12 The figure shows the statistical results of fermentation of parthenolide produced by 6 different genetically modified yeast strains. DETAILED DESCRIPTION

[0047] The present invention provides a yeast genetic engineering strain for producing parthenolide and a method for constructing the same. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0048] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0049] Example 1

[0050] Schematic diagram of the heme synthesis pathway in yeast Figure 1 As shown in the figure, the schematic diagram of the pathway for recombinant yeast to produce parthenolide is as follows Figure 2 Based on this, the preliminary construction and optimization method of heme regulation of the recombinant Saccharomyces cerevisiae strain producing parthenolide of the present invention are as follows:

[0051] 1. Obtaining a recombinant Saccharomyces cerevisiae strain producing parthenolide and a recombinant Saccharomyces cerevisiae strain producing costunolide

[0052] The strain SyBE_Sc07140055 producing parthenolide was provided by Xiao Wenhai's research group and is numbered ySYT055. The genetic modification contained in the recombinant Saccharomyces cerevisiae is: the ZWF1 gene is overexpressed on the basis of the strain ySYT0017. The strain is derived from the literature Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory.

[0053] 2. Obtaining recombinant yeast for producing costunolide

[0054] Strains SyBE_Sc07140053 and ySYT053 producing parthenolide

[0055] The gene modification contained in the recombinant Saccharomyces cerevisiae is: the ZWF1 gene is overexpressed on the basis of ySYT012. The strain is derived from the document Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory.

[0056] Among them, the SyBE_Sc07140053 (ySYT053) and SyBE_Sc07140055 (ySYT055) chassis strain information is shown in Table 1 below.

[0057] Table 1

[0058]

[0059] 3. Obtaining plasmid pYTQ090 and plasmid pYTQ092

[0060] 3.1 Obtaining plasmid pYTQ090: The PTS gene was cloned using template SyBE_Ec07140022. The authors designed to insert the parthenolide synthase PTS into the functional expression cassette WJ416k (the construction diagram is shown in Figure 4As shown in the figure, the functional expression cassette WJ416k was digested with BamHI enzyme, and a cut was obtained in the middle of TEF1-HXT7t to obtain a linearized vector. The upper and lower primers of the PTS gene were designed. The upper and lower primers added 20 bp homology arms at the two cuts of the vector to the two ends of TEF1 and HXT7t, respectively. The upper and lower primers added 20 bp homology arms at the two cuts of the vector to the two ends of TEF1 and HXT7t, respectively, with the help of a PCR instrument, and connected in vitro using a seamless cloning method. The connection system was introduced into DH5α competent cells together, and the transformants were selected for amplification and culture to obtain plasmids for sequencing. The plasmids with correct sequencing were used to construct mutants. The method for constructing mutants is the inversePCR method. That is, a pair of primers in opposite directions are set at the site where the mutation is required, and the length of the overlapping sequence of the two primers containing the mutation site is about 30 bp, and the primer annealing temperature is above 60°C. The amplified fragments were transformed into DH5α competent cells, and transformants were selected for amplification and culture to obtain plasmids for sequencing. Among the plasmids that were sequenced correctly, the plasmid in which the tyrosine at the 22nd position of the PTS of the parthenolide synthase was mutated to glycine was selected, and the TEF1-PTS was cloned using a PCR instrument. Y22G -HXT7t was cloned from the plasmid. Enzyme digestion and ligation into pRS426 vector resulted in plasmid pYTQ090 (construction diagram as shown in Figure 5 shown).

[0061] Among them, PTS Y22G The nucleotide sequence is shown below (SEQ ID NO: 1):

[0062]

[0063] PTS Y22G The amino acid sequence is shown below (SEQ ID NO: 3):

[0064] MDTSTSFPSLFLPTLCTILISGIIIKYVLIWNRSSMAAFNLPPSPPKLPIIGNIHHVFSKNVNQTLWKLSKKYGPVMLIDTGAKSFLVVSSSQMAMEVLKTHQEILSTRPSNEGTKRLSYNFSDIT FSPHGDHWRDMRKVFVNEFLGPKRAGWFNQVLRMEIKDVINNLSSNPLNTSINLNEMLLSLVYRVVCKFAFGKSYREEPFNGVTLKEMLDESMVVLAGSSADMFPTFGWILDKLYGWNDRLEKCFGN LDGFFEMIINEHLQSASETSEDEKDFVHSLVELSLKDPQFTKDYIKALLLNVLLGAIDTTFTTIVWAMSEIVKNTQVMQKLQTEIRSCIGRKEEVDATDLTNMAYLKMVIKETLRLHPPAPLLFPR ECPSHCKIGGYDVFPGTCVVMNGWGIARDPNVWKEIPNEFYPERFENFNIDFLGNHCEMIPFGAGRRSCPGMKSATSTIEFTLVNLLYWFDWEVPSGMNNQDLDMEEDGFLVIQKKSPLFLIPIKHI

[0065] 3.2 Obtaining plasmid pYTQ092

[0066] Using the genome of the Saccharomyces cerevisiae strain as a template, the TDH3p, TDH1t and HRD1 genes were cloned by PCR instrument, and the three fragments were assembled together using overlap extension PCR. The fragment TDH3p-HRD1-TDH1t was obtained, and the plasmid pYTQ090 was cut with restriction endonuclease SacI, and the fragment was seamlessly cloned into the plasmid pYTQ090. According to the transformation and sequencing procedures described above, plasmid pYTQ092 (pRS426, TEF1p-Y22G-HXT7t-TDH3p-HRD1-TDH1T) was obtained. The construction schematic diagram is shown in the figure. Figure 6 shown.

[0067] The nucleotide sequence of HRD1 is shown below (SEQ ID NO: 2):

[0068]

[0069] 4. Plasmids pYTQ093 and pYTQ094 containing the homology arm HEM2 gene with integration sites were obtained.

[0070] The TKL2 site on the yeast chromosome II and the Baco ori on the pE2μ plasmid of the chassis strain SyBE_Sc07140053 were selected as the integration sites. http: / / crispor.tefor.net / The gRNA sequence was designed based on the prediction results given by the website. The 500 bp fragments before and after the Bac ori site in the gRNA sequence binding region were used as homology arms to construct plasmid pYTQ093 with TEF1p-HEM2-HXT7t. The construction diagram is shown in the figure. Figure 7 As shown, plasmid pYTQ094 can be obtained by the same construction method. The construction diagram is shown in Fig. 9 shown.

[0071] 5. Obtaining gRNA plasmid for gene integration

[0072] pCRI2 and pZK028 were provided by Xiao Wenhai's laboratory, including:

[0073] The pCRI2 plasmid is a Cas9 plasmid with integrated genes. The sgRNA sequence on the plasmid can target the BAC ori site of the yeast μ plasmid with the protein transcribed by the guide Cas9. The construction diagram is shown in the figure. Figure 8 As shown;

[0074] pZK028 is used as the Cas9 plasmid for integrating the HEM2 gene, in which the sgRNA sequence is: This sgRNA sequence can target the TKL2 site on the yeast chromosome II with the protein transcribed by guiding Cas9. The construction schematic diagram is shown in Fig.10 shown.

[0075] 6. Construction of plasmid expressing heme gene

[0076] Considering the presence of uracil nutritional tags in the chassis strain, the authors designed to clone the heme genes HEM2, EHM3, and HEM12 (including HEM2: GeneID: 852842; HEM3: GeneID: 851322; HEM12: GeneID: 851617) from the yeast genome, digested the functional expression cassette pRS426 with XhoI enzyme, designed upper and lower primers for the HEM2, HEM3, and HEM12 genes, and added the 20 bp homology arms of the upper and lower primers to the two ends of TEF1 and HXT7t, respectively. EM2, HEM3, HEM12 and TEF1 were connected together to obtain TEF1-HEM2, TEF1-HEM3, TEF1-HEM12, and then TEF1-HEM2, TEF1-HEM3, TEF1-HEM12 and HXT7t were overlapped by extension PCR to obtain TEF1-HEM2-HXT7t, TEF1-HEM3-HXT7t, TEF1-HEM12-HXT7t. The upper and lower primers of these three fragments were added to the two ends of TEF1 and HXT7t at XhoI according to the homology arms of 20 bp each. The construction schematic diagram is shown in the figure. Figure 3 The seamless cloning method is used to connect in vitro, and the connection system is introduced into DH5α competent cells. The transformants are selected for amplification and culture to obtain plasmids for sequencing. The plasmids with correct sequencing are used for yeast transformation.

[0077] 7. Construction of a yeast strain that overexpresses the heme gene to produce parthenolide in the form of a plasmid

[0078] The pRS426 plasmids containing HEM2, HEM3, and HEM12 genes were transformed into SyBE_Sc07140055 by the lithium acetate method. After transformation, SC-URA solid plates (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 22 g / L, mixed amino acid powder lacking tryptophan, leucine, histidine, and uracil 2 g / L, 2% agar powder) were used for screening. The selected transformants were verified by 20 mm NaOH solution boiled bacteria PCR, and the verified single colonies were isolated and purified for fermentation detection.

[0079] The control strain was numbered SyBE_Sc07140055, and the experimental bacteria were numbered SyBE_Sc08150061 (yYTQ061) (overexpressing the HEM2 gene), SyBE_Sc08150062 (yYTQ062) (overexpressing the HEM3 gene), and SyBE_Sc08150063 (yYTQ063) (HEM12 gene).

[0080] 8. Fermentation assay to screen genes that can increase heme supply

[0081] Experimental materials: strains SyBE_Sc07140055 and SyBE_Sc08150061-SyBE_Sc08150063

[0082] Experimental methods:

[0083] Seed culture medium: SC-URA liquid medium (synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2g / L, tryptophan, leucine and histidine are supplemented in the form of 100* mother liquor); YPD medium (yeast extract powder 10g / L, peptone 20g / L, glucose 20g / L)

[0084] Fermentation medium: SC-URA liquid medium (synthetic yeast nitrogen source YNB 6.7g / L, glucose 40g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2g / L, tryptophan, leucine and histidine are supplemented in the form of 100* mother liquor). YPD medium (yeast extract powder 10g / L, peptone 20g / L, glucose 20g / L)

[0085] SyBE_Sc08150061-SyBE_Sc08150063 strains were inoculated into 5 mL SC-URA seed medium and cultured at 30 °C and 250 rpm for 14-16 h. The initial bacterial concentration OD 600 =0.2 were inoculated into 50 mL of fermentation medium and cultured at 30 °C and 220 rpm for 96 h. The cell density (OD 600 ) and the production of parthenolide.

[0086] SyBE_Sc07140055 strain was inoculated into 5 mL YPD seed medium and cultured at 30 °C and 250 rpm for 14-16 h. The initial bacterial concentration OD 600 =0.2 were inoculated into 50 mL of fermentation medium and cultured at 30 °C and 220 rpm for 96 h. The cell density (OD 600 ) and the production of parthenolide.

[0087] Quantitative detection method of parthenolide: Take 50ml of fermentation broth at the end of fermentation, centrifuge at 4000r to separate the bacteria, collect the upper organic phase, prepare the sample required for HPLC detection, and prepare the mother solution of parthenolide with methanol to obtain the standard curve. HPLC analysis was performed on Shimadzu SPD-20A and a BDS Hypersil C18 column (250mm×4.6mm, 5μm, ThermoScientific). Mobile phase A was 100% acetonitrile; mobile phase B was water + 0.1% formic acid. Isocratic elution, 50% A and 50% B, flow rate 1mL / min, 20min.

[0088] The experimental results are as follows Fig.11 As shown in the figure, among the four strains, after statistics on the production of parthenolide in each strain, it was found that the strain numbered SyBE_Sc08150061 had the highest production of 9.53 mg / L, which was about 43% higher than the control strain SyBE_Sc07140055. This indicates that the HEM2 gene can promote the production of heme in yeast, increase the catalytic activity of parthenolide synthase, and thus increase the production of parthenolide.

[0089] 9. Construction of strains with heme gene HEM2 integrated into yeast chromosomes

[0090] 9.1 Construction of strain SyBE_Sc08150094 with HEM2 gene integrated at 2μ locus

[0091] This project uses CRISPR / Cas9 technology to edit the genome of Saccharomyces cerevisiae. The screening tag of the gRNA expression plasmid is Ura3, and the tag needs to be recovered to achieve the purpose of multiple genome editing. The principle of Ura3 tag recovery is that yeast with Ura3 gene activity can convert 5-FOA into cytotoxic fluorodeoxyuridine, making yeast cells unable to grow in a culture medium containing 5-FOA. The specific experimental steps are as follows:

[0092] (1) Using pYTQ093 as a template, clone the fragment BAC up-TEF1p-HEM2-HXT7t-BAC down using a PCR instrument, transform the fragment together with pCRI2 into yeast using the lithium acetate transformation method, and after a single colony grows, perform the boiled bacteria verification as described above. The correct single colony is streaked on an SC plate containing 5-FOA and incubated upside down in a 30°C constant temperature incubator until a single colony grows;

[0093] (2) Pick a new single colony and streak it on a Sc-Ura plate and a YPD plate at the same time, and culture it upside down in a constant temperature incubator at 30°C;

[0094] (3) If no single colony can be grown on the Sc-Ura plate, but a single colony can be grown on the YPD plate, it indicates that the Ura3 tag has been successfully recovered.

[0095] 9.2 Construction of strain SyBE_Sc08150095 with HEM2 gene integrated at TKL2 locus

[0096] This project uses CRISPR / Cas9 technology to edit the genome of Saccharomyces cerevisiae. The screening tag of the gRNA expression plasmid is Ura3, and the tag needs to be recovered to achieve the purpose of multiple genome editing. The principle of Ura3 tag recovery is that yeast with Ura3 gene activity can convert 5-FOA into cytotoxic fluorodeoxyuridine, making yeast cells unable to grow in a culture medium containing 5-FOA. The specific experimental steps are as follows:

[0097] (1) Using pYTQ094 as a template, a fragment TKL2up-TEF1p-HEM2-HXT7t-TKL2down was cloned by PCR instrument, and the fragment was transformed into yeast SyBE_Sc07140053 together with pZK028 by lithium acetate ionization transformation method. After a single colony grew, the correct single colony was streaked on an SC plate containing 5-FOA and incubated upside down in a constant temperature incubator at 30°C until a single colony grew;

[0098] (2) Pick and verify the correct single colony and streak it on the SC plate containing 5-FOA, and incubate it upside down in a 30°C constant temperature incubator until a single colony grows;

[0099] (3) Pick a new single colony and streak it on a Sc-Ura plate and a YPD plate at the same time, and culture it upside down in a constant temperature incubator at 30°C;

[0100] (4) If no single colony can be grown on the Sc-Ura plate, but a single colony can be grown on the YPD plate, it indicates that the Ura3 tag has been successfully recovered.

[0101] 10. Construction of strains producing parthenolide:

[0102] 10.1 Construction of yYTQ099: Based on strain SyBE_Sc07140053, plasmid pYTQ090 was transformed into the strain according to the above-mentioned lithium acetate transformation method. After transformation, SC-URA solid plates (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 22 g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2 g / L, 2% agar powder) were used for screening. The selected transformants were verified by 20 mm NaOH solution boiled bacteria PCR, and the verified single colonies were isolated and purified for fermentation detection

[0103] 10.2 Construction of yYTQ100: Based on strain SyBE_Sc07140053, plasmid pYTQ092 was transformed into the strain according to the above-mentioned lithium acetate transformation method. After transformation, SC-URA solid plate (synthetic yeast nitrogen source YNB 6.7g / L, glucose 22g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2g / L, 2% agar powder) was used for screening. The selected transformants were verified by 20mmNaOH solution boiling PCR, and the verified single colonies were isolated and purified for fermentation detection.

[0104] 11. Construction of a strain that produces parthenolide with heme gene integrated into its genome

[0105] (1) Construction of a strain (yYTQ103) that produces parthenolide and integrates the HEM2 gene at the 2μ site

[0106] Based on strain SyBE_Sc08150094, plasmid pYTQ090 was transformed into the strain according to the above-mentioned lithium acetate transformation method. After transformation, SC-URA solid plate (synthetic yeast nitrogen source YNB 6.7g / L, glucose 22g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2g / L, 2% agar powder) was used for screening. The selected transformants were verified by 20mmNaOH solution boiling PCR, and the verified single colonies were isolated and purified for fermentation detection.

[0107] (2) Construction of a strain (yYTQ104) that integrates the HEM2 gene at the 2μ site and overexpresses the HRD1 gene to produce parthenolide

[0108] Based on strain SyBE_Sc08150094, plasmid pYTQ092 was transformed into the strain according to the above-mentioned lithium acetate transformation method. After transformation, SC-URA solid plate (synthetic yeast nitrogen source YNB 6.7g / L, glucose 22g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2g / L, 2% agar powder) was used for screening. The selected transformants were verified by 20mmNaOH solution boiling PCR, and the verified single colonies were isolated and purified for fermentation detection.

[0109] (3) Construction of a strain (yYTQ107) that produces parthenolide and integrates the HEM2 gene at the TKL2 locus

[0110] The plasmid pYTQ090 was transformed into the strain SyBE_Sc08150095 by the lithium acetate transformation method. After transformation, SC-URA solid plates (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 22 g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2 g / L, 2% agar powder) were used for screening. The selected transformants were verified by 20 mm NaOH solution boiled bacteria PCR. The verified single colonies were isolated and purified for fermentation detection.

[0111] (4) Construction of a strain (yYTQ108) that integrates the HEM2 gene at the TKL2 locus and overexpresses the HRD1 gene to produce parthenolide

[0112] Based on the strain SyBE_Sc08150095, the plasmid pYTQ092 was transformed into the strain according to the lithium acetate transformation method. After transformation, SC-URA solid plate (synthetic yeast nitrogen source YNB 6.7g / L, glucose 22g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2g / L, 2% agar powder) was used for screening. The selected transformants were verified by 20mmNaOH solution boiling PCR, and the verified correct single colonies were isolated and purified for fermentation detection.

[0113] 12. Fermentation of strains producing parthenolide with HEM2 gene integrated into their genome

[0114] The fermentation culture method and detection method of the strain with heme gene integrated into the genome to produce parthenolide are consistent with the fermentation culture method and detection method of strains SyBE_Sc07140055 and SyBE_Sc08150061-SyBE_Sc08150063. Fig.12 Shown

[0115] The fermentation results showed that when the HEM2 gene was integrated at the 2μ site, it did not have a positive effect on increasing the production of parthenolide. However, when the HEM2 gene was integrated at the TKL2 site, it could improve the strain's ability to produce parthenolide, and when the HRD1 gene was overexpressed, the production of parthenolide reached 27.08 mg / L, which was much higher than the shake flask production level of parthenolide reported in the literature.

[0116] The above are only preferred embodiments 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of at least one of the following (1) to (4) in increasing the production of parthenolide in recombinant Saccharomyces cerevisiae: (1) Gene encoding HEM2; (2) Mutant PTS of parthenolide synthase PTS Y22G genes; (3) the gene encoding HRD1; (4) TKL2 locus as a gene integration site; The recombinant Saccharomyces cerevisiae is SyBE_Sc07140053.

2. An expression cassette, characterized in that Includes at least one of expression cassettes 1 to 4 The expression cassette 1 includes TEF1-PTS Y22G -HXT7t; The expression cassette 2 includes TDH3p-HRD1-TDH1t; The expression cassette 3 includes TEF1p-Y22G-HXT7t-TDH3p-HRD1-TDH1T; The expression cassette 4 includes TEF1p-HEM2-HXT7t.

3. The expression cassette according to claim 2, characterized in that The expression cassette 4 is expressed at the TKL2 site.

4. An expression vector, characterized in that It comprises a backbone vector and the expression cassette according to claim 2 or 3.

5. A host cell, characterized in that Comprising the expression cassette of claim 2 or 3 and / or the expression vector of claim 4; The host cell includes Saccharomyces cerevisiae.

6. A recombinant strain, characterized in that The expression cassette according to claim 2 or 3 and / or the expression vector according to claim 4 is transformed or introduced into the chassis strain.

7. The recombinant strain according to claim 5, characterized in that The chassis strain is recombinant Saccharomyces cerevisiae SyBE_Sc07140053, and the coding gene of HEM2 is integrated into the TKL2 site of the chassis strain.

8. The method for constructing the recombinant strain according to claim 6 or 7, characterized in that: The method comprises genetically modifying the chassis strain, wherein the modification comprises the modification shown in ①, or further comprises the modification shown in ② and / or ③ on the basis of ①: ①, transfer the coding gene of HEM2; ②, overexpression of the mutant PTS of parthenolide synthase PTS Y22G genes; ③, overexpression of the gene encoding HRD1; Preferably, the gene encoding HEM2 is integrated into the TKL2 site of the chassis strain.

9. Use of at least one of the following Ⅰ to Ⅴ in the synthesis of parthenolide: Ⅰ. The expression cassette according to claim 2 or 3; II. The expression vector according to claim 4; III. The host cell according to claim 5; IV. The recombinant strain according to claim 6 or 7; V. The recombinant strain constructed by the construction method according to claim 8.

10. A method for synthesizing parthenolide, characterized in that: The method comprises culturing the recombinant strain according to claim 6 or 7 or the recombinant strain constructed by the construction method according to claim 8 to obtain parthenolide.

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