Parthenolide synthetase variant, parthenolide high-yield engineering bacterium and application

Abstract

The mutant capable of improving the catalytic capacity of the parthenolide synthetase is obtained through computer-aided directed evolution and fermentation detection of enzymes, the mutant and an HRD1 gene are co-expressed and used for synthesis of parthenolide, and experimental results show that the parthenolide synthetase containing the mutant Y22G has the highest synthesis yield of parthenolide, and the yield of parthenolide is high. The mutant Y22G and HRD1 are expressed together, and the yield of parthenolide continues to be increased; compared with an existing reported strain for producing parthenolide, the recombinant saccharomyces cerevisiae strain is higher in yield, is the highest strain for producing parthenolide through shake flask fermentation at present, and has a wide industrial application prospect.

Description

Technical Field

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

[0002] Parthenolide is a sesquiterpene lactone and a direct precursor of the anti-glioblastoma drug ACT001. Currently, the parthenolide extracted from dried parthenolide leaves and magnolia root bark cannot meet the growing market demand. According to literature reports, the shake flask yield of the recombinant yeast strain producing costunolide reached 239 mg / L. However, after the introduction of parthenolide synthase into the strain producing costunolide, the shake flask yield of parthenolide was only 8.75 mg / L, and the accumulation of costunolide was 16 mg / L. The reason may be that the heterologous parthenolide synthase and yeast have large genotype differences; and Saccharomyces cerevisiae is an important eukaryotic cell factory. When constructing heterologous metabolic or synthetic pathways, the expression problem of heterologous genes must be overcome first.

[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 variant of parthenolide synthase, an engineered bacterium with high production of parthenolide and its application.

[0005] The present invention provides a parthenolide synthase variant, which comprises: 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 invention firstly optimizes the amino acid sequence of the parthenolide synthase shown in SEQ ID NO:1 by means of computer-assisted directed evolution of an enzyme, and 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 test results show that after the tyrosine Y at position 22 is mutated, the yield of parthenolide in the parthenolide synthase is significantly increased compared with that in the non-mutated parthenolide synthase (p≤0.01), and the yield of parthenolide is higher than that of 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 production of parthenolide in the Y22G and Y22D mutants was higher than that in other mutations, among which the Y22G mutant had the highest production of parthenolide.

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

[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 endoplasmic reticulum stress-related molecular chaperone (HRD1) in series. The test results show that the yield 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 can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. Nucleic acid can 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 acid can be linear or circular in topology. Nucleic acid can be a part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The DNA can also be obtained by reverse transcription of RNA, which is not limited by the present invention.

[0017] In the present invention, the nucleic acid may be optimized or unoptimized, 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, secondary structure of mRNA, 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 starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc.

[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, in the screening process of parthenolide synthase, the WJ416k functional box is used to optimize the parthenolide synthase, wherein the WJ416k functional box 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 2micron 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, which comprises at least one of the nucleic acid of the present invention or the expression cassette of the present invention and a vector backbone.

[0023] Further, 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: adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, or lentivirus vectors, etc. The phage vectors include phagemids and auxiliary 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 vector includes but is not limited to pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vector includes but is 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] 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 that is essential for the expression of an operably connected coding gene in a specific host organism. In this specification, "plasmid" and "vector" can sometimes be interchanged and used, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or only potential genome insert.

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

[0026] Furthermore, the sources of the host cells include plants, animals, bacteria, fungi, bacteriophages 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 is used as an intermediate host for the construction, preservation, reproduction or other functions of the recombinant vector described in 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 electrotransformation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, and the like.

[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 by using at least one of the following A) to F):

[0037] 1), 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 enzymes and fermentation detection to obtain a mutant capable of improving the catalytic ability of parthenolide synthase, and the mutant is co-expressed with an HRD1 gene for the synthesis of parthenolide. Experimental results show that the parthenolide synthase containing the mutant Y22G has the highest synthesis yield of parthenolide, and the yield of parthenolide is further improved by co-expressing the mutant Y22G and HRD1. The recombinant brewer's yeast strain of the present invention has a higher yield than the existing reported strain for producing parthenolide, is the highest strain currently producing parthenolide by shake flask fermentation, and has 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 The result diagram showing the production of parthenolide by fermentation of the screened parthenolide synthase mutants;

[0048] Figure 5 Show YTQ 416K RFP;

[0049] Figure 6 Fluorescence numerical diagram of the superior mutant of parthenolide synthase;

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

[0051] Figure 8 1 and 2 show plasmid maps, wherein A is a map of pYTQ090, and B is a map of plasmid pYTQ092;

[0052] Fig. 9 The figure shows 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 production of parthenolide and applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all 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.

[0054] Amino acid sequence of parthenolide synthase:

[0055]

[0056] Nucleotide sequence of endoplasmic reticulum stress-related molecular chaperone (HRD1):

[0057]

[0058]

[0059] The SyBE_Sc07140053 chassis strain information is shown in Table 1 of the literature "Production of Plant SesquiterpeneLactone Parthenolide in the Yeast Cell Factory", and the specific genotype is shown in Table 1:

[0060] Table 1. Gene information of SyBE_Sc07140053 chassis strain

[0061]

[0062] In Table 1, ySYT053 is the SyBE_Sc07140053 chassis strain of the present invention.

[0063] In the present invention, in the significance analysis, ** represents p<0.01; * represents p<0.05.

[0064] 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 in conjunction with the embodiments:

[0065] Example 1 Construction of an engineered bacterium with high production of parthenolide

[0066] The present invention uses a strain of Saccharomyces cerevisiae SyBE_Sc07140053 producing costunolide as a base bacteria, transforms a parthenolide synthase mutant into the yeast, screens out mutants with better catalytic activity through fermentation yield detection, and then co-expresses the mutants with the HRD1 gene to achieve the effect of increasing yield.

[0067] The preliminary construction and optimization method for improving the production of the recombinant Saccharomyces cerevisiae strain by screening the parthenolide synthase mutants is as follows:

[0068] 1. Selection of parthenolide synthase mutants:

[0069] 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).

[0070] 2. Obtaining a recombinant Saccharomyces cerevisiae strain containing a parthenolide synthase mutant that produces parthenolide:

[0071] The strain number of the recombinant yeast chassis bacteria is SyBE_Sc07140053. The genetic modification contained in the recombinant cerevisiae yeast is: on the basis of ySYT012, the ZWF1 gene is overexpressed (the ySYT012 strain is derived from the Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory, and the ZWF1 gene is a costunolide synthesis gene); on this basis, the present invention transfers a plasmid containing a WJ416k functional box into the chassis bacteria SyBE_Sc07140053, and the WJ 416K functional box is as follows Figure 2 As shown, the target gene was inserted between the TEE1p promoter and the HXT7t terminator.

[0072] 3. Construction of mutant plasmids and acquisition of yeast strains containing mutants that produce parthenolide

[0073] (1) Construction of mutant plasmid: Considering the presence of a uracil nutritional label in the chassis strain, the present invention is 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 between TEF1 and HXT7t to obtain a linearized vector, design upper and lower primers of the PTS gene, and add 20 bp of homologous arms at the two incisions of the vector to the two ends of TEF1 and HXT7t respectively with the upper and lower primers. The two homologous arms at the two incisions of the vector are added 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 is introduced into DH5α competent cells together, and transformants are selected for amplification and culture to obtain plasmids for sequencing. The plasmid pYTQ001 (plasmid map is shown in FIG. 1 ) that is sequenced correctly is Figure 3The mutants were constructed by inverse PCR. That is, a pair of primers in opposite directions were set at the site where the mutation was required, the length of the overlapping sequence of the two primers containing the mutation site was about 30 bp, the primer annealing temperature was above 60°C, and plasmids containing PTSs of different mutation sites were obtained.

[0074] (2) Construction of yeast strains containing mutant plasmids for producing parthenolide: Plasmids containing different mutation sites PTS were transformed into the original base bacterium SyBE_Sc07140053 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. The verified single colonies were isolated and purified for fermentation testing. The strains containing different mutation sites PTS to be fermented and tested are shown below:

[0075] The control strain (containing only wild-type parthenolide synthase) is numbered SyBE_Sc08150001, and the experimental bacteria include:

[0076] SyBE_Sc08150020(T3M),SyBE_Sc08150021(L10M),SyBE_Sc08150022(T14F),SyBE_Sc08150023(T14S),SyBE_Sc08150024(I20F),Sy BE_Sc08150025(S21A),SyBE_Sc08150026(Y22F),SyBE_Sc08150027(I24F),SyBE_Sc08150028(Y27W),SyBE_Sc08150029(V28F),SyB E_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);

[0077] and mutant test bacteria with Y22 as the only 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 (Y22A), SyBE_Sc08150049 (Y22B), SyBE_Sc08150050 (Y22C), SyBE_Sc08150051 (Y22D), SyBE_Sc08150052 (Y22E), SyBE_Sc08150053 (Y22F), SyBE_Sc08150054 (Y22G), SyBE_Sc08150055 (Y22M), SyBE_Sc08150056 (Y22S), SyBE_Sc08150057 (Y22V), SyBE_Sc08150058 (Y22E), SyBE_Sc08150059 (Y22F), SyBE_Sc08150060 (Y22H), SyBE_Sc08150061 (Y22H), SyBE_Sc08150062 (Y22C), SyBE_Sc08150063 (Y22R), SyBE_Sc08150064 (Y22G), SyBE_Sc08150065 (Y22M), SyBE_Sc08150066 (Y22S), SyBE_Sc08150067 48(Y22I),SyBE_Sc08150049(Y22W),SyBE_Sc08150050(Y22T),SyBE_Sc08150051(Y22L),SyBE_Sc08150052(Y22D),SyBE_S c08150053(Y22K), SyBE_Sc08150054(Y22Q), SyBE_Sc08150055(Y22N), SyBE_Sc08150056(Y22E), SyBE_Sc08150057(Y22A);

[0078] 4. Experimental bacteria fermentation test for mutant screening

[0079] Experimental materials: strains SyBE_Sc08150001, SyBE_Sc08150020-SyBE_Sc08150057.

[0080] Experimental methods:

[0081] Seed culture medium: SC-URA liquid medium (synthetic yeast nitrogen source 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 are supplemented in the form of 100× mother solution);

[0082] 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× mother liquor).

[0083] The above strains were inoculated into 5 mL of 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.

[0084] 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.

[0085] Experimental results: Among the 35 strains, after statistics on the production of parthenolide of each strain, it was found that the strain numbered SyBE_Sc08150044 (Y22G) had the highest production, reaching 20.58 mg / L ( Figure 4 ).

[0086] 5. Fluorescence intensity analysis of excellent mutants:

[0087] Experimental materials: strain 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);

[0088] Experimental methods:

[0089] The RFP gene was cloned using a PCR instrument using SyBE_Ec07140028 as a template, and the RFP gene was inserted into the functional expression cassette WJ416k. The functional expression cassette WJ416k was digested with BamHI enzyme, and a cut was made 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 20 bp of homologous arms at the two cuts of the vector to the two ends of TEF1 and HXT7t, respectively, and the upper primer carried 6 bases of the SalI site. The seamless cloning method was used for in vitro connection, and the connection system was introduced into DH5α competent cells together. The transformants were selected for amplification and culture, and the plasmids were sequenced. The plasmids with correct sequencing were used as the functional cassette YTQ416K-RFP (TEF1p-RFP-HXT7t) (the structure of YTQ416K-RFP is as shown in Figure 5As shown, the corresponding experimental material strain name is SyBE_Sc08150086 (TEF1p-RFP-HXT7t). After obtaining YTQ 416k-RFP, the functional box YTQ 416k RFP was digested with SalI enzyme, and the PTS and Y22G genes were connected to the YTQ 416kRFP functional box according to the above method (the strain name in the corresponding experimental material is SyBE_Sc08150088 (TEF1p-Y22G-RFP-HXT7t). And introduced into DH5α competent cells according to the same method as above, and the transformants were selected for amplification culture to obtain plasmids for sequencing. The plasmids with correct sequencing were used for yeast transformation, and the DH5α strain SyBE_Sc08150085 (WJ 416k) containing empty WJ 416k and the DH5α strain SyBE_Sc08150087 without mutation of the parthenolide synthase PTS were used as references.

[0090] Fluorescence value determination: The transformed single colony was inoculated with SC-U liquid medium. After culturing the primary seed for 20-24 hours and the secondary seed for 48 hours, the fluorescence intensity of the cells per OD 600 was detected using an ELISA reader, and the fluorescence intensity was detected at an excitation wavelength of 553 nm and an emission wavelength of 583 nm.

[0091] 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 ).

[0092] 6. Construction of strains for optimizing production by co-expressing superior mutants of parthenolide synthase and HRD1 gene

[0093] 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 ), construct plasmid pYTQ090 (pRS426, TEF1p-Y22G-HXT7t) (plasmid map as shown in Figure 8 ).

[0094] The plasmid was transformed into the yeast strain SyBE_Sc07140053 to obtain the yeast strain SyBE_Sc08150099 (SyBE_Sc07140053; pYTQ090). The genome of the Saccharomyces cerevisiae strain was used as a template, and the TDH3p (promoter), TDH1t (terminator) 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 the restriction endonuclease SacI, and the fragment was seamlessly cloned into the plasmid pYTQ090. According to the transformation, sequencing and other processes described above, the plasmid pYTQ092 (pRS426, TEF1p-Y22G-HXT7t-TDH3p-HRD1-TDH1T) was obtained. The plasmid map is shown in FIG. Figure 8 As shown in B. The plasmid was transformed into yeast strain SyBE_Sc07140053 to obtain yeast strain SyBE_Sc08150100 (SyBE_Sc07140053; pYTQ092).

[0095] 7. Fermentation verification of strains for optimizing production by co-expressing superior mutants of parthenolide synthase and molecular chaperone genes

[0096] Experimental materials: strains SyBE_Sc08150044, SyBE_Sc08150099, SyBE_Sc08150100 Experimental methods:

[0097] Seed culture medium: SC-URA liquid medium (synthetic yeast nitrogen source 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 are supplemented in the form of 100× mother solution);

[0098] 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× mother liquor).

[0099] The above strains were inoculated into 5 mL of seed culture medium and cultured at 30°C and 250 rpm for 14 to 16 h. The initial bacterial concentration OD600 was inoculated into 50 mL of fermentation medium at 0.2, and cultured at 30°C and 220 rpm for 96 h. The bacterial density (OD600) and the production of parthenolide at the end of the fermentation were monitored.

[0100] 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.

[0101] Experimental results: Among the three strains, after statistics on the production of parthenolide in each strain, it was found that the strain numbered SyBE_Sc081500100 had the highest production, while the production of SyBE_Sc081500099 was lower. It may be that after the multi-copy expression of Y22G, the misfolded proteins produced increased the pressure of the endoplasmic reticulum. By co-expressing Y22G and HRD1 genes on multi-copy plasmids, the overexpression of HRD1 gene can reduce the degradation of misfolded proteins and reduce the pressure of the endoplasmic reticulum. Its production reached 23.4mg / L ( Fig. 9 , where Costunolide is costunolide). It is the yeast strain with the highest production of parthenolide in shake flasks.

[0102] 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 include: The parthenolide synthase having an amino acid sequence as shown in SEQ ID NO: 1 has at least one of the following mutations: 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 described in claim 1 and a molecular chaperone associated with endoplasmic reticulum stress.

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

2.

4. A nucleic acid, characterized in that Including at least one of the following i) to iii): 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 The method comprises at least one of a promoter, a terminator, a replicon and / or a selection marker and the nucleic acid according to claim 4.

6. A recombinant vector, characterized in that It 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 bottom plate bacteria are derived from Saccharomyces cerevisiae.

9. Application 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 of 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 using at least one of the following A) to F): 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 of claim 5; V), the recombinant vector according to claim 6; VI), the host cell according to claim 7 or 8.

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