Parthenolide synthase variants, engineered bacteria with high parthenolide production, and applications thereof

Abstract

The present invention utilizes computer-assisted directed evolution of an enzyme and fermentation testing to obtain a mutant capable of improving the catalytic ability of parthenolide synthase. The mutant is co-expressed with the HRD1 gene for the synthesis of parthenolide. Experimental results show that the parthenolide synthase containing the mutant Y22G has the highest yield for the synthesis of parthenolide, and the co-expression of the mutant Y22G and HRD1 further increases the yield of parthenolide. The recombinant Saccharomyces cerevisiae strain of the present invention has a higher yield than existing reported strains for producing parthenolide, and is currently the strain with the highest yield for producing parthenolide by shake flask fermentation, thus having broad industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a parthenolide synthase variant, an engineered bacterium with high parthenolide production, and applications thereof. Background Art

[0002] Parthenolide, a sesquiterpene lactone, is the direct precursor of the anti-glioblastoma drug ACT001. Current parthenolide extractions from dried feverfew leaves and magnolia root bark cannot meet the growing market demand. Literature reports indicate that recombinant yeast strains producing costunolide have achieved shake-flask yields of 239 mg / L. However, after introducing parthenolide synthase into the costunolide-producing strain, the shake-flask yield was only 8.75 mg / L, while the accumulated costunolide level was 16 mg / L. This may be due to significant genotypic differences between the heterologous parthenolide synthase and the yeast. Since Saccharomyces cerevisiae is a key eukaryotic cell factory, the expression of heterologous genes must be overcome when constructing heterologous metabolic or synthetic pathways.

[0003] Therefore, in order to overcome the low production of parthenolide caused by low expression and activity of heterologous genes in yeast, and to further increase the production of parthenolide, it is particularly necessary to develop new engineered strains with high production of parthenolide. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a parthenolide synthase variant, an engineered bacterium with high parthenolide production, and applications thereof.

[0005] The present invention provides a parthenolide synthase variant, comprising: a parthenolide synthase having an amino acid sequence as shown in SEQ ID NO: 1 and having at least one of the following mutations:

[0006] The tyrosine Y at position 22 is mutated to glycine G, aspartic acid D, methionine M, serine S, valine V, isoleucine I, tryptophan W, threonine T, phenylalanine F, leucine L or alanine A.

[0007] The present invention first uses a computer-assisted directed enzyme evolution method to optimize the amino acid sequence of the parthenolide synthase shown in SEQ ID NO: 1. Specifically, the optimized sites include but are not limited to: T3, L10, T14 I20 S21, Y22, I24, Y27, V28, L29, I3, N32, Q64, M96, N112, S200, V220, L432, and Y467. The experimental results show that after the tyrosine Y at position 22 is mutated, the parthenolide production of the parthenolide synthase is significantly increased compared with that of the non-mutated one (p≤0.01), and the parthenolide production is higher than that of the other mutations.

[0008] Subsequently, further optimization was carried out on the tyrosine Y at position 22, and the following mutations were performed: Y22H, Y22C, Y22P, Y22R, Y22G, Y22M, Y22S, Y22V, Y22I, Y22W, Y22T, Y22L, Y22D, Y22K, Y22Q, Y22N, Y22E and Y22A; the experimental results showed that the Y22G and Y22D mutants had higher parthenolide production than other mutations, among which the Y22G mutant had the highest parthenolide production.

[0009] In the present invention, the means of computer-assisted directed evolution of enzymes include but are not limited to PSSM matrix, which is not limited in the present invention.

[0010] The present invention provides a protein combination, which includes the parthenolide synthase variant of the present invention and an endoplasmic reticulum stress-related molecular chaperone (HRD1);

[0011] Furthermore, the nucleotide encoding the endoplasmic reticulum stress-related molecular chaperone is shown in SEQ ID NO: 2.

[0012] Based on the optimization of parthenolide synthase, the present invention expresses the endoplasmic reticulum stress-related molecular chaperone (HRD1) in tandem. The experimental results show that the production of parthenolide is further improved.

[0013] The present invention provides a nucleic acid comprising at least one of the following i) to iii):

[0014] i), a nucleic acid encoding the parthenolide synthase variant of the present invention;

[0015] ii), a nucleic acid encoding the protein combination of the present invention.

[0016] The nucleic acid encoding the recombinant antigen described in the present invention may be DNA, RNA, cDNA, or PNA. In embodiments of the present invention, the nucleic acid is in the form of DNA. Such DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. Nucleic acids may include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids may be linear or circular in topology. Nucleic acids may be part of a vector (e.g., an expression or cloning vector) or a fragment. The nucleic acid may be obtained directly from a natural source or may be prepared using recombinant, enzymatic, or chemical techniques. The DNA may also be obtained by reverse transcription of RNA, which is not a limitation of the present invention.

[0017] In the present invention, the nucleic acid may be optimized or not, and the optimization includes but is not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0018] The present invention provides a transcription unit, which refers to a DNA sequence beginning with a promoter and ending with a terminator. The promoter and terminator may also be flanked or interposed with regulatory segments. These regulatory segments may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, an origin of replication, a nucleic acid restriction site, and a homologous recombination site, such as a promoter enhancer and a poly(A) signal, operably linked to the nucleic acid sequence.

[0019] The present invention provides an expression cassette comprising at least one of a promoter, a terminator, a replicon and / or a selection marker and the nucleic acid of the present invention.

[0020] In some specific embodiments of the present invention, during the screening process of parthenolide synthase, the WJ416k functional cassette is used to optimize the parthenolide synthase, wherein the WJ416k functional cassette includes a TEF1 promoter, an HXT7t stop codon, a KanR promoter and a KanR resistance screening marker, a Ura3 promoter and a Ura3 screening marker, and a 2 micron origin replication start site; the parthenolide synthase gene is inserted between the TEEF1 promoter and the HXT7t stop codon;

[0021] In other specific embodiments of the present invention, the parthenolide synthase variant and the endoplasmic reticulum stress-related molecular chaperone (HRD1) are co-expressed; wherein the parthenolide synthase variant and the endoplasmic reticulum stress-related molecular chaperone (HRD1) have different promoters and terminators; specifically, the promoter of the endoplasmic reticulum stress-related molecular chaperone (HRD1) is TDH3p, and the terminator is TDH1t.

[0022] The present invention provides a recombinant vector comprising at least one of the nucleic acid of the present invention or the expression cassette of the present invention and a vector backbone.

[0023] Furthermore, the sources of the vector backbone described in the present invention include plants, animals, bacteria, fungi, phages or viruses, which are not limited by the present invention. The viral vectors include: adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, or lentiviral vectors. The phage vectors include phagemids and helper vectors, and the phagemids include but are not limited to pBluescript II-KS (+), pcomb3XSS, pCANTAB5E or pKK233.3. The animals include mammals and non-mammals, and the mammalian expression vectors include but are not limited to pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vectors include, but are not limited to, pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vectors include, but are not limited to, pYES2, pYES3, pYES6, pAUR23, etc. In a specific embodiment of the present invention, a plasmid capable of integrating the WJ416k functional cassette is used.

[0024] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence or element necessary for the expression of an operably linked coding gene in a specific host organism. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are the most commonly used vector form. However, the present invention is intended to include other forms of expression vectors that function equivalently and are known or will become known in the art, including but not limited to plasmids, phage particles, viral vectors and / or only potential genomic inserts.

[0025] The present invention provides a host cell, the genome of which is integrated with the nucleic acid of the present invention or the expression cassette of the present invention, or is transfected or transformed with the recombinant vector of the present invention.

[0026] Furthermore, the sources of the host cells include plants, animals, bacteria, fungi, phages or viruses, which are not limited in the present invention; the present invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, so that the transformed host cells have the ability to replicate protein-encoding vectors or express desired proteins.

[0027] In some specific embodiments of the present invention, the host cell is derived from bacteria or fungi; specifically, the bacteria include Escherichia coli and Bacillus subtilis, etc., and the Escherichia coli includes but is not limited to Escherichia coli DH5α, which serves as an intermediate host for the construction, preservation, propagation or other functions of the recombinant vector of the present invention; the fungi include yeast, algae and / or mold; specifically, the yeast includes but is not limited to Saccharomyces cerevisiae, Saccharomyces lipolytica, Kluyveromyces and / or Pichia pastoris, etc.; in a specific embodiment of the present invention, the genotype of the chassis strain of the host cell is as shown in Table 1, which is Saccharomyces cerevisiae CEN.PK2-1D containing the costunolide synthesis gene ZWF1 and the WJ416k functional box.

[0028] In the present invention, the transformation methods include chemical transformation and electroporation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection methods include adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0029] The present invention provides the use of at least one of the following I) to VI) in the synthesis of parthenolide:

[0030] 1), the parthenolide synthase variant of the present invention;

[0031] II), the protein combination of the present invention;

[0032] III), the nucleic acid of the present invention;

[0033] IV), the expression cassette of the present invention;

[0034] V), the recombinant vector of the present invention;

[0035] VI), the host cell of the present invention.

[0036] The present invention provides a method for synthesizing parthenolide, characterized in that the method comprises synthesizing parthenolide using at least one of the following A) to F):

[0037] I), the parthenolide synthase variant of the present invention;

[0038] II), the protein combination of the present invention;

[0039] III), the nucleic acid of the present invention;

[0040] IV), the expression cassette of the present invention;

[0041] V), the recombinant vector of the present invention;

[0042] VI), the host cell of the present invention.

[0043] The present invention utilizes computer-assisted directed evolution of an enzyme and fermentation testing to obtain a mutant capable of improving the catalytic ability of parthenolide synthase. The mutant is co-expressed with the HRD1 gene for the synthesis of parthenolide. Experimental results show that the parthenolide synthase containing the mutant Y22G has the highest yield for the synthesis of parthenolide, and the co-expression of the mutant Y22G and HRD1 further increases the yield of parthenolide. The recombinant Saccharomyces cerevisiae strain of the present invention has a higher yield than existing reported strains for producing parthenolide, and is currently the strain with the highest yield for producing parthenolide by shake flask fermentation, thus having broad industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The principle of using PSSM matrix to screen mutants is shown;

[0045] Figure 2 Show WJ 416K function box;

[0046] Figure 3 A diagram showing plasmid pYTQ001;

[0047] Figure 4 A diagram showing the results of fermentation of parthenolide production by the screened parthenolide synthase mutants;

[0048] Figure 5 Show YTQ 416K RFP;

[0049] Figure 6 Fluorescence numerical graph of superior mutants of parthenolide synthase;

[0050] Figure 7 shows a map of the multicopy plasmid pRS426;

[0051] Figure 8 Shown are plasmid maps, wherein A is a map of pYTQ090, and B is a map of plasmid pYTQ092;

[0052] Figure 9 Graph showing the fermentation results of the strain co-expressing Y22G and HRD1 genes. DETAILED DESCRIPTION

[0053] The present invention provides variants of parthenolide synthase, engineered bacteria with high parthenolide production, and applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be 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 herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0054] Amino acid sequence of parthenolide synthase: MDTSTSFPSLFLPTLCTILISYIIIKYVLIWNRSSMAAFNLPPSPPKLPIIGNIHHVFSKNVNQTLWKLSKKYGPVMLIDTGAKSFLVVSSSQMAMEVLKTHQEILSTRPSNEGTKRLSYNFSDITFSPHGDHWRDMRKVFVNEFLGPKRAGWFNQVLRMEIKDVINNLSSNPLNTSINLNEMLLSLVYRVVCKFAFGKSYREEPFNGVTLKEMLDESMVVLAGSSADMFPTFGWILDKLYGWNDRL EKCFGNLDGFFEMIINEHLQSASETSEDEKDFVHSLVELSLKDPQFTKDYIKALLLNVLLGAIDTTFTTIVWAMSEIVKNTQVMQKLQTEIRSCIGRKEEVDATDLTNMAYLKMVIKETLRLHPPAPLLFP RECPSHCKIGGYDVFPGTCVVMNGWGIARDPNVWKEIPNEFYPERFENFNIDFLGNHCEMIPFGAGRRSCPGMKSATSTIEFTLVNLLYWFDWEVPSGMNNQDLDMEEDGFLVIQKKSPLFLIPIKHI (SEQ ID NO:1);

[0055]

[0056] The SyBE_Sc07140053 chassis strain information is shown in Table 1 of the literature "Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory". The specific genotype is shown in Table 1 below:

[0057] Table 1. Gene information of SyBE_Sc07140053 chassis strain

[0058]

[0059] In Table 1, ySYT053 is the SyBE_Sc07140053 chassis strain of the present invention.

[0060] In the present invention, in the significance analysis, ** represents p < 0.01; * represents p < 0.05.

[0061] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below with reference to the following examples:

[0062] Example 1 Construction of an engineered bacterium with high production of parthenolide

[0063] The present invention uses a costunolide-producing Saccharomyces cerevisiae strain SyBE_Sc07140053 as the base strain, transforms a parthenolide synthase mutant into the yeast, screens out mutants with better catalytic activity through fermentation yield detection, and then co-expresses them with the HRD1 gene to achieve the effect of increasing yield.

[0064] The preliminary construction and optimization method for improving the production of a recombinant Saccharomyces cerevisiae strain by screening for a feverfew lactone synthase mutant is as follows:

[0065] 1. Selection of parthenolide synthase mutants:

[0066] According to the PSSM matrix ( http: / / possum.erc.monash.edu / ) predicted amino acid score results, the original amino acid is mutated to an amino acid with a higher score than the wild-type amino acid (the principle of using PSSM matrix to screen mutants is as follows Figure 1 shown).

[0067] 2. Obtaining a recombinant Saccharomyces cerevisiae strain containing a parthenolide synthase mutant that produces parthenolide:

[0068] The recombinant yeast strain is numbered SyBE_Sc07140053. The genetic modifications included in this recombinant Saccharomyces cerevisiae include overexpression of the ZWF1 gene based on ySYT012 (the ySYT012 strain is derived from the Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory; the ZWF1 gene is a costunolide biosynthesis gene). Based on this, the present invention transferred a plasmid containing the WJ416k functional cassette into the strain to obtain the base strain SyBE_Sc07140053. The WJ 416K functional cassette is as follows: Figure 2 As shown, the target gene was inserted between the TEE1p promoter and the HXT7t terminator.

[0069] 3. Construction of mutant plasmids and acquisition of yeast strains containing mutants that produce parthenolide

[0070] (1) Construction of mutant plasmid: Considering the presence of uracil nutritional label in the chassis strain, the present invention designed to insert the parthenolide synthase PTS into the functional expression cassette WJ416k, digest the functional expression cassette WJ416k with BamHI enzyme, obtain an incision in the middle of TEF1-HXT7t to obtain a linearized vector, design the upper and lower primers of the PTS gene, and add the 20bp homologous arms at the two incisions of the vector to the two ends of TEF1 and HXT7t respectively. The upper and lower primers add the 20bp homologous arms at the two incisions of the vector to the two ends of TEF1 and HXT7t respectively with the help of a PCR instrument, connect them in vitro using the seamless cloning method, and introduce the connection system into DH5α competent cells together. The transformants are selected for amplification and culture to obtain the plasmid for sequencing. The plasmid pYTQ001 (plasmid map as shown in the figure) that has been sequenced correctly Figure 3 Mutants were constructed using the inverse PCR method. This involves setting a pair of primers in opposite directions at the desired mutation site. The overlapping sequence of the two primers, encompassing the mutation site, is approximately 30 bp long. The primer annealing temperature is above 60°C, resulting in plasmids containing PTSs at different mutation sites.

[0071] (2) Construction of yeast strains containing mutant plasmids for producing parthenolide: Plasmids containing different mutation sites PTS were transformed into the original base strain SyBE_Sc07140053 using 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 PCR in 20 mM NaOH solution. The correct single colonies were isolated and purified for fermentation testing. The strains containing different mutation sites PTS to be tested for fermentation are shown below:

[0072] The control strain (containing only wild-type parthenolide synthase) is numbered SyBE_Sc08150001. The experimental strains include:

[0073] SyBE_Sc08150020 (T3M), SyBE_Sc08150021 (L10M), SyBE_Sc08150022 (T14F), SyBE_Sc08150023 (T14S), SyBE_Sc08150024 (I20F), SyBE_Sc08150025 (S21A), SyBE_Sc08150026 (Y22F), SyBE_Sc08150027 (I24F), SyBE_Sc08150028 (Y27W), SyBE_Sc08150029 (V28F), SyBE_Sc08150030 (L29F), SyBE_Sc08150031 (I30F), SyBE_Sc08150032 (N32K), SyBE_Sc08150033 (Q64R), SyBE_Sc08150034 (M96K), SyBE_Sc08150035 (N112P), SyBE_Sc08150036 (S200R), SyBE_Sc08150037 (V220E), SyBE_Sc08150038 (L432K), SyBE_Sc08150039 (Y467H);

[0074] And the mutant test bacteria with Y22 as the main optimization: SyBE_Sc08150040 (Y22H), SyBE_Sc08150041 (Y22C), SyBE_Sc08150042 (Y22P), SyBE_Sc08150043 (Y22R), SyBE_Sc08150044 (Y22G), SyBE_Sc08150045 (Y22M), SyBE_Sc08150046 (Y22S), SyBE_Sc08150047 (Y22V), SyBE_Sc08150048 (Y22I), SyBE_Sc08150049 (Y22W), SyBE_Sc08150050 (Y22T), SyBE_Sc08150051 (Y22L), SyBE_Sc08150052 (Y22D), SyBE_Sc08150053 (Y22K), SyBE_Sc0815 0054 (Y22Q), SyBE_Sc08150055 (Y22N), SyBE_Sc08150056 (Y22E), SyBE_Sc08150057 (Y22A);

[0075] 4. Experimental bacterial fermentation test for mutant screening

[0076] Experimental materials: strains SyBE_Sc08150001, SyBE_Sc08150020- SyBE_Sc08150057.

[0077] Experimental methods:

[0078] Seed culture medium: SC-URA liquid medium (synthetic yeast nitrogen base YNB 6.7 g / L, glucose 20 g / L, mixed amino acid powder deficient in tryptophan, leucine, histidine, and uracil 2 g / L, tryptophan, leucine, and histidine supplemented in the form of 100× stock solution);

[0079] Fermentation medium: SC-URA liquid medium (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 40 g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2 g / L, tryptophan, leucine and histidine are supplemented in the form of 100× stock solution).

[0080] The above strains were inoculated into 5 mL seed culture 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.

[0081] Quantitative determination of parthenolide: 50 mL of fermentation broth at the end of the fermentation was centrifuged at 4000 r / min to separate the bacterial cells. The upper organic phase was collected and used as the sample for HPLC analysis. A mother solution of parthenolide was prepared with methanol to prepare the standard curve. HPLC analysis was performed using a Shimadzu SPD-20A column and a BDS Hypersil C18 column (250 mm × 4.6 mm, 5 μm, ThermoScientific). Mobile phase A was 100% acetonitrile; mobile phase B was water + 0.1% formic acid. Isocratic elution was performed with 50% A and 50% B at a flow rate of 1 mL / min over 20 minutes.

[0082] Experimental results: Among the 35 strains, after statistical analysis of the parthenolide production of each strain, it was found that the strain numbered SyBE_Sc08150044 (Y22G) had the highest production, reaching 20.58 mg / L ( Figure 4 ).

[0083] 5. Fluorescence intensity analysis of superior mutants:

[0084] Experimental materials: strains SyBE_Sc08150085 (WJ 416k), SyBE_Sc08150086 (TEF1p- RFP-HXT7t), SyBE_Sc08150087 (TEF1p-PTS-RFP-HXT7t), SyBE_Sc08150088 (TEF1p-Y22G-RFP-HXT7t), SyBE_Ec07140028 (ygg416-GAL1p-HaGAS1-RFP-TDH2t);

[0085] Experimental methods:

[0086] Using SyBE_Ec07140028 as a template, the RFP gene was cloned using a PCR instrument and inserted into the functional expression cassette WJ416k. The functional expression cassette WJ416k was digested with BamHI enzyme to obtain a cut in the middle of TEF1-HXT7t to obtain a linearized vector. The upper and lower primers of the RFP gene were designed. The upper and lower primers added 20bp of homology arms at the two cuts of the vector to the two ends of TEF1 and HXT7t, respectively, and the upper primer contained 6 bases of the SalI site. The seamless cloning method was used for in vitro ligation, and the ligation system was introduced into DH5α competent cells. The transformants were selected for amplification and culture, and the plasmid was obtained for sequencing. The plasmid with correct sequencing was used as the functional cassette YTQ416K-RFP (TEF1p-RFP-HXT7t) (YTQ416K-RFP structure is shown in Figure 2). Figure 5As shown, the corresponding experimental material strain is named SyBE_Sc08150086 (TEF1p-RFP-HXT7t). After obtaining YTQ 416k-RFP, the functional cassette YTQ 416k RFP was digested with SalI enzyme, and the PTS and Y22G genes were ligated into the YTQ 416k RFP functional cassette according to the above method (the corresponding experimental material strain is named SyBE_Sc08150088 (TEF1p-Y22G-RFP-HXT7t). The transfection was then transformed into DH5α competent cells according to the same method as above. Transformants were selected for amplification and plasmid extraction for sequencing. Plasmids that were sequenced correctly were used for yeast transformation. The DH5α strain SyBE_Sc08150085 (WJ 416k) containing a null WJ 416k gene and the DH5α strain SyBE_Sc08150087 without the mutated parthenolide synthase PTS were used as references.

[0087] Fluorescence measurement: Inoculate a single colony grown from the transformation into SC-U liquid medium. After culturing the primary seed for 20–24 hours and the secondary seed for 48 hours, measure the fluorescence intensity of the cells at OD600 using a microplate reader, using an excitation wavelength of 553 nm and an emission wavelength of 583 nm.

[0088] Experimental results: After comparing the fluorescence intensity, the fluorescence intensity of SyBE_Sc08150088 was about 1.6 times higher than that of SyBE_Sc08150087, indicating that the reason why Y22G improved the catalytic ability of parthenolide synthase was that the expression of parthenolide synthase was enhanced ( Figure 6 ).

[0089] 6. Construction of strains for optimizing production by co-expressing superior mutants of parthenolide synthase and HRD1 gene

[0090] After obtaining the parthenolide synthase mutant Y22G with higher catalytic activity, the present invention continues to express Y22G in multiple copies, that is, using the pRS426 plasmid to express the Y22G gene in multiple copies (the plasmid is constructed as shown in FIG. Figure 7 ), and the plasmid pYTQ090 (pRS426, TEF1p-Y22G-HXT7t) was constructed (plasmid map as shown in Figure 8 (as shown in A in the figure).

[0091] The plasmid was transformed into the yeast strain SyBE_Sc07140053 to obtain the yeast strain SyBE_Sc08150099 (SyBE_Sc07140053; pYTQ090). Using the genome of Saccharomyces cerevisiae as a template, the TDH3p (promoter), TDH1t (terminator), and HRD1 genes were cloned by PCR. The three fragments were assembled using overlap extension PCR. The resulting fragment, TDH3p-HRD1-TDH1t, was digested with the restriction endonuclease SacI and seamlessly ligated into the plasmid pYTQ090. Following the transformation and sequencing procedures described above, plasmid pYTQ092 (pRS426, TEF1p-Y22G-HXT7t-TDH3p-HRD1-TDH1T) was obtained. The plasmid map is shown in Figure 2. Figure 8 As shown in Figure B. The plasmid was transformed into the yeast strain SyBE_Sc07140053 to obtain the yeast strain SyBE_Sc08150100 (SyBE_Sc07140053; pYTQ092).

[0092] 7. Fermentation verification of strains for optimizing production by co-expressing superior mutants of parthenolide synthase with molecular chaperone genes

[0093] Experimental materials: strains SyBE_Sc08150044, SyBE_Sc08150099, SyBE_Sc08150100

[0094] Experimental methods:

[0095] Seed culture medium: SC-URA liquid medium (synthetic yeast nitrogen base YNB 6.7 g / L, glucose 20 g / L, mixed amino acid powder lacking tryptophan, leucine, histidine, and uracil 2 g / L, tryptophan, leucine, and histidine supplemented in the form of 100× stock solution);

[0096] Fermentation medium: SC-URA liquid medium (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 40 g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2 g / L, tryptophan, leucine and histidine are supplemented in the form of 100× stock solution).

[0097] The above strains were inoculated into 5 mL of seed culture medium and cultured at 30°C and 250 rpm for 14-16 h. They were then inoculated into 50 mL of fermentation medium with an initial cell concentration of OD600 = 0.2 and cultured at 30°C and 220 rpm for 96 h. The cell density (OD600) and the production of parthenolide at the end of the fermentation were monitored.

[0098] Quantitative determination of parthenolide: 50 mL of fermentation broth at the end of the fermentation was centrifuged at 4000 r / min to separate the bacterial cells. The upper organic phase was collected and used as the sample for HPLC analysis. A mother solution of parthenolide was prepared in methanol to prepare the calibration curve. HPLC analysis was performed using a Shimadzu SPD-20A column and a BDS Hypersil C18 column (250 mm × 4.6 mm, 5 μm, ThermoScientific). Mobile phase A was 100% acetonitrile; mobile phase B was water + 0.1% formic acid. Isocratic elution was performed with 50% A and 50% B at a flow rate of 1 mL / min for 20 minutes.

[0099] Experimental results: After statistical analysis of the parthenolide production of each of the three strains, it was found that the strain numbered SyBE_Sc081500100 had the highest production, while the production of SyBE_Sc081500099 was lower. This may be because after the multi-copy expression of Y22G, the misfolded proteins produced increased the pressure on the endoplasmic reticulum. By co-expressing Y22G and the HRD1 gene on a multi-copy plasmid, the overexpression of the HRD1 gene can reduce the degradation of misfolded proteins and reduce the pressure on the endoplasmic reticulum. Its production reached 23.4 mg / L ( Figure 9 , where costunolide is costunolide). This is the yeast strain with the highest production of parthenolide in shake flasks.

[0100] 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. A variant of parthenolide synthase, characterized in that The amino acid sequence of the parthenolide synthase variant is based on the amino acid sequence shown in SEQ ID NO: 1, The tyrosine at position 22 is mutated to glycine, aspartic acid, methionine, serine, valine, isoleucine, tryptophan, threonine, phenylalanine, leucine or alanine.

2. A protein combination, characterized in that The invention comprises the parthenolide synthase variant according to claim 1 and a molecular chaperone associated with endoplasmic reticulum stress.

3. The protein combination according to claim 2, characterized in that The nucleotide sequence encoding the endoplasmic reticulum stress-related molecular chaperone is shown in SEQ ID NO:

2.

4. A nucleic acid, characterized in that Including one of the following i) to ii): i), a nucleic acid encoding the parthenolide synthase variant according to claim 1; ii), a nucleic acid encoding the protein combination according to claim 2 or 3.

5. An expression cassette, characterized in that It comprises a promoter, a terminator and the nucleic acid according to claim 4.

6. A recombinant vector, characterized in that The method comprises at least one of the nucleic acid according to claim 4 or the expression cassette according to claim 5 and a vector backbone.

7. A host cell, characterized in that Genome integration of the nucleic acid according to claim 4 or the expression cassette according to claim 5, or transfection or transformation of the recombinant vector according to claim 6.

8. The host cell according to claim 7, characterized in that The host cell is derived from Saccharomyces cerevisiae.

9. Use of at least one of the following I) to VI) in the synthesis of parthenolide: 1), the parthenolide synthase variant according to claim 1; II), the protein combination according to claim 2 or 3; III), the nucleic acid according to claim 4; IV), the expression cassette according to claim 5; V), the recombinant vector according to claim 6; VI), the host cell according to claim 7 or 8.

10. A method for synthesizing parthenolide, characterized in that: The method comprises synthesizing parthenolide by utilizing at least one of the following methods I) to VI): 1), the parthenolide synthase variant according to claim 1; II), the protein combination according to claim 2 or 3; III), the nucleic acid according to claim 4; IV), the expression cassette according to claim 5; V), the recombinant vector according to claim 6; VI), the host cell according to claim 7 or 8.

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