Solution-promoting expression tag and application thereof

Abstract

The invention discloses a dissolution-promoting expression tag and application thereof, and belongs to the technical field of gene engineering. The solubilizing expression tag is thioredoxin Trx1 or Trx2 from yeast, and the amino acid sequence of the solubilizing expression tag is as shown in SEQ ID NO. 1 or SEQ ID NO. 2. Compared with other solubilizing tags from biological sources, the solubilizing expression tag provided by the invention can better promote correct folding of the exogenous fusion protein in the yeast, improve the soluble expression quantity and overcome the condition that the soluble expression of the heterologous protein in a yeast expression system is not ideal. The solubilizing expression tag is applied to heterologous expression protein of a yeast expression system, expression of heterologous protein is provided, the yield of target metabolites is further increased, and the solubilizing expression tag has a good industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering, and in particular to a solubilizing expression tag suitable for heterologous protein expression in a yeast expression system and an application thereof. Background Art

[0002] With the development of molecular biology and synthetic biology, the construction of heterologous pathways in Saccharomyces cerevisiae to achieve de novo synthesis of natural products has attracted much attention. However, due to differences in the intracellular microenvironment, heterologous proteins in biosynthetic pathways often have problems with correct folding, which affects their soluble expression and thus affects the synthesis efficiency of natural products (Soluble Expression of Disulfide Bond Containing Proteins FGF15 and FGF19 in the Cytoplasm of Escherichia coli. PLOS One, 2014, 9(1): e85890).

[0003] Protein fusion tags have the ability to reduce protein degradation, increase protein expression, promote protein folding and reduce recombinant protein degradation (Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein Science, 2006, 15 (1): 182-189). Common fusion tags include small ubiquitin-related modifiers (SUMO), thioredoxin (TRX), maltose binding protein (MBP), NUS A and glutathione S-transferase (GST). Studies have fused the SUMO tag to the N-terminus of PylB from M. acetivorans, and significantly improved the solubility of PylB in Escherichia coli through intracellular refolding of inclusion bodies (Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway. Nature Communications, 2021, 12 (1)). Some studies have fused SUMO and MBP tags with taxadiene synthase from Taxus cuspidate to improve its soluble expression in Saccharomyces cerevisiae, thereby increasing the yield of its catalytic product taxadiene from 0.80 mg / L to 3.55 mg / L and 3.45 mg / L, respectively (Improving solubility and copy number of taxadiene synthase to enhance the titer of taxadiene in Yarrowialipolytica. Synthetic and Systems Biotechnology, 2023, 8(2): 331-338).

[0004] Except for SMT3, a homolog of SUMO in Saccharomyces cerevisiae, which has been reported to be used as a lytic expression tag (Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside. International Journal of Molecular Sciences, 2020, 21(16): 5752), most of the other commonly used lytic expression tags are from prokaryotes. At present, lytic expression tags are mostly used to promote the expression of exogenous proteins in prokaryotes such as Escherichia coli, and there are still few tags that have been successfully applied in Saccharomyces cerevisiae. In addition, we found during the research process that prokaryotic lytic expression tags are not always suitable for the lytic expression of exogenous proteins in Saccharomyces cerevisiae.

[0005] Therefore, in order to promote the soluble expression of heterologous proteins in Saccharomyces cerevisiae and thereby improve the synthesis efficiency of natural products from heterologous pathways, it is urgent to find more solubility-promoting expression tags suitable for the soluble expression of foreign proteins in Saccharomyces cerevisiae. Summary of the invention

[0006] The purpose of the present invention is to provide a solubility-promoting expression tag suitable for soluble expression of foreign proteins in yeast, which is applied to a yeast expression system to improve the expression of foreign proteins.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention obtains a candidate lytic expression tag by searching for the endogenous thioredoxin Trx family of Saccharomyces cerevisiae on the Saccharomyces cerevisiae genome website (https: / / www.yeastgenome.org / ), and aligning its sequence with the currently commonly used lytic tag TrxA from Escherichia coli, and further verifies its lytic expression effect by fusion expression with exogenous genes, and finally obtains two proteins Trx1 and Trx2 with lytic expression activity.

[0009] Specifically, the amino acid sequence of Trx1 protein is shown in SEQ ID NO.1, which consists of 103 amino acids, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0010] The amino acid sequence of Trx2 protein is shown in SEQ ID NO.2, which consists of 104 amino acids, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0011] Therefore, the present invention provides a use of yeast-derived thioredoxin as a soluble expression tag, wherein the amino acid sequence of the thioredoxin is shown in SEQ ID NO. 1 or SEQ ID NO. 2. The soluble expression tag is fused with an exogenous gene to promote its soluble expression in a heterologous host.

[0012] Furthermore, the lytic expression tag is a lytic expression tag of a yeast expression system. The yeast may be, but is not limited to, Saccharomyces cerevisiae. The lytic expression tag is used to allow the foreign protein to be correctly folded in Saccharomyces cerevisiae, thereby improving the expression of the foreign protein.

[0013] Furthermore, the application includes: inserting the coding sequence of the soluble expression tag into the upstream of the exogenous protein encoding gene in the gene expression box, and fusing it with the exogenous protein encoding gene in the host cell to improve the soluble expression of the exogenous protein.

[0014] The gene expression cassette includes a promoter, a foreign protein encoding gene and a terminator, and the coding sequence of the lytic expression tag is inserted downstream of the promoter. The gene expression cassette can be integrated into the host cell chromosome for expression, or can be expressed on a free plasmid introduced into the host cell.

[0015] Furthermore, the coding sequence of the lytic expression tag is inserted into the 5' end of the start codon ATG of the foreign protein encoding gene as a lytic tag.

[0016] Furthermore, the host cell is Saccharomyces cerevisiae.

[0017] Furthermore, the coding sequence of the lytic expression tag is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0018] Furthermore, the application includes: using yeast as a starting bacterium, integrating a gene expression cassette inserted with a lytic expression tag coding sequence into a yeast chromosome via an integration plasmid, thereby obtaining a genetically engineered bacterium that solublely expresses foreign proteins;

[0019] Alternatively, yeast is used as a starting bacterium, and a recombinant expression plasmid containing a gene expression cassette inserted with a lytic expression tag coding sequence is introduced to obtain a genetically engineered bacterium that solublely expresses foreign proteins.

[0020] Furthermore, the integration plasmid is a pUMRI series assembly tool plasmid. The promoter is P GAL1 , P GAL10The terminator is T CYC1 、T ADH1 .

[0021] In the present invention, the lytic expression tag is applied to construct a genetically engineered strain with a heterologous biosynthetic pathway, and then the constructed genetically engineered bacteria are fermented and cultured to extract the target product from the fermentation product. The present invention constructs the lytic expression tag upstream of the exogenous expression gene to promote the soluble expression of the exogenous protein, so that it participates in the biosynthetic pathway of the target metabolite, thereby improving the synthesis efficiency of the target product of the heterologous pathway.

[0022] For example, the genetically engineered bacteria are genetically engineered bacteria that produce lutein, bisabolene, canthaxanthin or astaxanthin. Preferably, the exogenous protein is carotenoid β-hydroxylase from Arabidopsis thaliana, bisabolene synthase from Abies grandis, and β-carotene ketolase from Haematococcus pluvialis.

[0023] Specifically, when constructing a genetically engineered strain producing lutein, the lycopene-producing saccharomyces cerevisiae engineered strain YCF01 is used as the starting strain, and the genes Gal4M9 (Gene ID: 855828), CrtYB (AY177204), At-LUT2 (AAB53336.1), FD3 (Gene ID: 817297), RFNR1 (Gene ID: 825887), LUT1 (NP_190881.2), and CYP97A3 (AAG50718.1) are inserted into the genome or introduced using a recombinant expression plasmid, and the Gal4 of saccharomyces cerevisiae itself is knocked out. The solubility-promoting expression tag is constructed upstream of the CYP97A3 gene, thereby increasing the soluble expression of the key exogenous enzyme CYP97A3 in the lutein synthesis pathway, thereby increasing the lutein production.

[0024] The engineered strain YCF01 is a publicly available material, and its construction method can be found in the literature (Heterologous biosynthesis of lutein in S.cerevisiae enabled by temporospatial pathway control, Metabolic Engineering, 2021, 67: 19-28).

[0025] When constructing a genetically engineered strain producing bisabolene, the yeast engineering strain BY4742-C04 is used as the starting strain, and the genes ERG20 (GeneID: 853272) and AgBIS (AF006195) are inserted into the genome or introduced using a recombinant expression plasmid. The solubility-promoting expression tag is constructed upstream of the AgBIS gene, and the soluble expression of the key exogenous enzyme bisabolene synthase in the bisabolene synthesis pathway is increased, thereby increasing the yield of bisabolene.

[0026] The bisabolene-producing engineered strain BY4742-C04 with an enhanced MVA pathway was constructed, which is publicly available material. Its construction method is described in the literature (Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering, Metabolic Engineering, 2015, 30: 69-78).

[0027] When constructing genetically engineered bacteria for Cantharidin or astaxanthin, the β-carotene-producing yeast strain Ycarot-02 is used as the starting bacteria, and the gene OBKTM29 (KP866870) or OBKTM29 (KP866870) and OcrtZM1 (KP866869) are introduced into the genome by using a recombinant expression plasmid. The solubility-promoting expression tag is constructed upstream of the OBKTM29 gene, which promotes the soluble expression of the key exogenous enzyme β-carotene ketolase in the Cantharidin synthesis pathway, thereby increasing the yield of Cantharidin, and further increasing the yield of the downstream product astaxanthin.

[0028] The β-carotene-producing engineered strain Ycarot-02 is a publicly available material, and its construction method can be found in the literature (Construction of Canthaxanthin-Producing Yeast by Combining Spatiotemporal Regulation and Pleiotropic Drug Resistance Engineering, ACS Synthetic Biology, 2022, 11: 325-333).

[0029] The present invention has the following beneficial effects:

[0030] The present invention provides a new use of yeast-derived thioredoxin Trx1 or Trx2 as a lytic expression tag. Compared with lytic tags from other biological sources, the lytic expression tag provided by the present invention can better promote the correct folding of exogenous fusion proteins in yeast, increase the soluble expression amount, and overcome the unsatisfactory soluble expression of heterologous proteins in yeast expression systems. The present invention applies the lytic expression tag to heterologous protein expression in a yeast expression system, improves the expression of exogenous proteins, and then increases the yield of target metabolites, which has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the plasmid map of pUMRI-Trx1.

[0032] Figure 2 PCR verification results for the construction of pUMRI-Trx1 plasmid.

[0033] Figure 3 This is the sequencing result of pUMRI-Trx1 plasmid.

[0034] Figure 4 This is the plasmid map of pUMRI-Trx2.

[0035] Figure 5 PCR verification results for the construction of pUMRI-Trx2 plasmid.

[0036] Figure 6 This is the sequencing result of pUMRI-Trx2 plasmid.

[0037] Figure 7 The results of liquid chromatography analysis of lutein production before and after the application of different solubilizing tags.

[0038] Figure 8 These are the gas chromatography analysis results of bisabolene produced before and after the application of different solubilizing labels.

[0039] Fig. 9 These are the results of liquid chromatography analysis of canthaxanthin before and after the application of different solubilizing labels.

[0040] Fig.10 The results of liquid chromatography analysis of astaxanthin production before and after the application of Trx1 solubilizing tag. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] The primers in the following examples were synthesized by Beijing Qingke Biotechnology Co., Ltd. The PCR reaction kit was purchased from Dalian Bao Biotechnology Co., Ltd. The PCR purification kit was purchased from Suzhou Yuheng Biotechnology Co., Ltd. TOROIVD One Step Fusion CloningMix was purchased from Toyobo.

[0044] The construction method of the lycopene-producing Saccharomyces cerevisiae engineered strain YCF01 used in the following examples is described in the literature (Heterologous biosynthesis of lutein in S.cerevisiae enabled by temporospatial pathway control, Metabolic Engineering, 2021, 67: 19-28);

[0045] The construction method of yeast engineering strain BY4742-C04 is described in the literature (Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering, Metabolic Engineering, 2015, 30: 69-78);

[0046] For the construction method of the β-carotene engineering strain Ycarot-02, please refer to the literature (Construction of Canthaxanthin-Producing Yeast by Combining Spatiotemporal Regulation and Pleiotropic Drug Resistance Engineering, ACS Synthetic Biology, 2022, 11: 325-333).

[0047] Example 1: PCR amplification of Trx1 gene

[0048] The base sequence of the Trx1 gene is shown in SEQ ID NO.3, and has a length of 309 bp.

[0049] (a) PCR primer design and reaction conditions

[0050] A set of primers was designed based on the Trx1 gene sequence and the DNA sequence of pUMRI:

[0051] Forward Primer:

[0052] 5'-GAAAAAACCCCGGATCCGCGATGGTTACTCAATTCAAAAC-3';

[0053] Reverse Primer:

[0054] 5'-TTAGATGAAGATGAAAACATAGCATTAGCAGCAATGGCTT-3';

[0055] The PCR reaction system was: 10 μL of 5× PrimeSTAR Buffer, 4 μL of dNTP Mixture, 15 pmol of upstream and downstream primers, 150 ng of template Saccharomyces cerevisiae BY4741 genome, 0.5 L of PrimeSTAR HSDNA Polymerase, and sterile water to make up the system to 50 μL.

[0056] The PCR reaction conditions were as follows: first stage: 98°C, 3 min; second stage: 98°C, 10 s; 55°C, 10 s; 72°C, 30 s; 30 cycles in total; third stage: 68°C, 5 min; fourth stage: 16°C, 1 min;

[0057] (b) PCR product processing: After the PCR amplification product was verified by 1% agarose gel electrophoresis, it was cleaned by a PCR purification kit, and the product was the obtained Trx1 gene fragment.

[0058] Example 2: PCR amplification of Trx2 gene

[0059] The base sequence of the Trx2 gene is shown in SEQ ID NO.4, and has a length of 312 bp.

[0060] (a) PCR primer design and reaction conditions

[0061] A set of primers was designed based on the Trx2 gene sequence and the DNA sequence of pUMRI:

[0062] Forward Primer:

[0063] 5'-GAAAAAACCCCGGATCCGCGATGGTCACTCAATTAAAATC-3';

[0064] Reverse Primer:

[0065] 5'-TTAGATGAAGATGAAAACATTACGTTGGAAGCAATAGCTT-3'.

[0066] The PCR reaction was the same as in Example 1.

[0067] The PCR reaction conditions were the same as those in Example 1.

[0068] (b) PCR product treatment: The PCR amplification product was treated in the same manner as in Example 1, and the product was the obtained Trx2 gene fragment.

[0069] Example 3: Construction of expression cassette of Trx1 or Trx2 and foreign protein expression gene

[0070] (1) Design of pUMRI vector PCR

[0071] The vector was amplified by PCR, and different primers were designed according to the sequences of the vector and exogenous enzyme, see Table 1.

[0072] Table 1. Primers required for constructing vectors carrying Trx1 or Trx2

[0073]

[0074] The PUMR1 used in the backbone of the plasmid construction is a publicly available material, and its construction method is described in the literature (Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering, Metabolic Engineering, 2015, 30: 69-78).

[0075] The PCR reaction system was: 5× PrimeSTAR Buffer 10 μL, dNTP Mixture 4 μL, 15 pmol of upstream and downstream primers, and templates were pUMRI-T ADH1 -LUT1-P GAL10 -P GAL1 -CYP97A3-T CYC1 , pUMRI-T CYC1 -AgBIS-ERG20-P GAL1 -P GAL10 -T ADH1 , pUMRI-T CYC1 -OcrtZM1-P GAL1 -P GAL10 -OBKTM29-T ADH1 150ng, PrimeSTAR HS DNA Polymerase 0.5L, and sterile water to make up the system to 50μL.

[0076] pUMRI-T CYC1 -AgBIS-ERG20-P GAL1 -P GAL10 -T ADH1 The construction of the AgBIS-ERG20 fusion protein is to insert the gene encoding the PUMR1 skeleton.

[0077] The PCR reaction conditions were the same as in Example 1.

[0078] (2) PCR product processing:

[0079] The PCR product was processed as in Example 1.

[0080] The purified product was digested with restriction enzyme Dpn I.

[0081] The digestion system is: 17 μL of purified product, 2 μL of QuickCut Buffer, and 1 μL of QuickCut TM Dpn I 1 μL.

[0082] Digestion conditions: 37°C, 2h.

[0083] (3) Vector construction and amplification

[0084] (a) Vector construction

[0085] The purified product obtained in Example 1 or 2 and the digested product obtained in Example 3 were subjected to Gibson analysis using TOROIVD OneStep Fusion Cloning Mix.

[0086] The Gibson reaction system is: 2×One Step Fusion Cloning Mix 5μL, linearized vector 50-200ng, insert fragment 10-200ng, ddH2O to make up to 10μL.

[0087] The Gibson reaction conditions are: 50°C, 5-60 min.

[0088] (b) Vector amplification

[0089] The competent E. coli cells BL21 were taken out from -80℃ and revived on ice for 3-5min. ADH1 -LUT1-P GAL10 -P GAL1 -Trx1-CYP97A3-T CYC1 or pUMRI-T ADH1 -LUT1-P GAL10 -PGAL1 -Trx2-CYP97A3-T CYC1 After the competent cells were transferred, the BL21 cells were placed in an ice bath for 15 minutes. The BL21 cells were then heat-shocked at 42°C for 90 seconds and then continued to be placed in an ice bath for 5 minutes. Then 1 mL of LB medium was added to the BL21 cells of each system, and the cells were revived in a shaker at 37°C for 40 minutes. Most of the supernatant was discarded after centrifugation, and an appropriate amount of bacterial liquid was applied to an LB plate with kanamycin added for screening, and cultured in a 37°C incubator for 12 hours.

[0090] (c) Sequence verification of vector

[0091] Pick a single colony from the resistance plate that has been cultured for 12 hours for PCR verification. Amplify and culture the single colony whose verification band meets the target length, and take a sample to Beijing Qingke Biotechnology for sequencing. The bacterial solution with the correct sequencing is cultured in a test tube with kanamycin for 12 hours, and the plasmid is extracted and stored.

[0092] pUMRI-T ADH1 -LUT1-P GAL10 -P GAL1 -Trx1-CYP97A3-T CYC1 The plasmid map, single colony PCR verification, and sequencing results are as follows Figure 1-3 shown.

[0093] pUMRI-T ADH1 -LUT1-P GAL10 -P GAL1 -Trx2-CYP97A3-T CYC1 The plasmid map, single colony PCR verification, and sequencing results are as follows Figure 4-6 shown.

[0094] At the same time, the vector pUMRI-T was constructed ADH1 -LUT1-P GAL10 -P GAL1 -SMT3-CYP97A3

[0095] -T CYC1 , pUMRI-T ADH1 -LUT1-P GAL10 -P GAL1 -NusA-CYP97A3-T CYC1 , pUMRI-T ADH1 -LUT1-P GAL10 -P GAL1 -MBP-CYP97A3-T CYC1 , pUMRI-T ADH1 -LUT1

[0096] -PGAL10 -P GAL1 -GST-CYP97A3-T CYC1 As a control, the gene sequence information of SMT3 can be found in Gene ID: 852122, the gene sequence information of NusA can be found in Gene ID: 947682, the gene sequence information of MBP can be found in Gene ID: 105302711, and the gene sequence information of GST can be found in Gene ID: 914142.

[0097] Example 4: Application and effect evaluation of Trx1 and Trx2 lysis-promoting expression tags in lutein synthesis

[0098] (1) Construction of lutein-producing strains carrying Trx1 or Trx2 lytic expression tags

[0099] Using the plasmid obtained in Example 3 as a template, HS DNA polymerase primers amplified T with homology arms ADH1 -LUT1-P GAL10 -P GAL1 -Trx1-CYP97A3-T CYC1 or T ADH1 -LUT1-P GAL10 -P GAL1 -Trx2-CYP97A3-T CYC1 The expression cassette was concentrated by ethanol precipitation and used as the integration donor.

[0100] Primers for amplifying expression cassette:

[0101] Forward primer: 5′-ACAATAAGGACATTTCTTGGTGACAGATTCGTTAAGTTTCCTGAATTGGAGCGACCTCA-3′;

[0102] Reverse primer: 5′-CCTTCACCCTCATCAATAATTTTCAATTCGTCGTTTCACCCTTCGAGCGTCCCAAAACC-3′.

[0103] Using the LiAc / SS carrier DNA / PEG method (High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. Nature Protocols, 2007, 2(1):31-34), T ADH1 -LUT1-P GAL10 -P GAL1 -Trx1-CYP97A3-TCYC1 or T ADH1 -LUT1-P GAL10 -P GAL1 -Trx2-CYP97A3-T CYC1 The expression cassettes were respectively integrated into the chromosome of the engineering strain of lycopene-producing Saccharomyces cerevisiae YCF01, and the genes Gal4M9, CrtYB, At-LUT2, FD3, and tRFNR1 were inserted into the genome or introduced using a recombinant expression plasmid to knock out the Gal4 of the engineering strain of Saccharomyces cerevisiae itself.

[0104] (2) Fermentation culture of lutein-producing strains and extraction and analysis of products

[0105] A single colony of the engineered bacteria was picked from the streaked plate and inoculated into 5 mL of YPD medium for about 16 hours. The strain was then inoculated into 50 mL of YPD liquid medium to an initial OD of 600nm The fermentation was carried out in a constant temperature shaker at 220 rpm and 30°C for 96 h.

[0106] Collect 0.2mL yeast fermentation broth into a 2mL centrifuge tube, centrifuge at 12000rpm for 2min, discard the supernatant, then wash twice with 1mL distilled water, centrifuge and discard the supernatant, add about 0.5mL volume of grinding beads (half 0.1mm and half 0.5mm zirconium oxide beads) to the centrifuge tube, add 200μL of acetone to the centrifuge tube, and resuspend the bacteria. Place the centrifuge tube containing beads and bacteria in a fully automatic sample rapid grinder, 65HZ, and grind for 5min. After grinding, add 800μL of acetone to the centrifuge tube, mix thoroughly, and place it in ultrasound for 5min. After ultrasound, place the centrifuge tube in a 4℃ centrifuge at 12000rpm for 2min, and collect the supernatant into a new 2mL centrifuge tube. Filter the extract with a 0.22μm organic filter head and wait for HPLC detection.

[0107] The following conditions were used to detect various carotenoids and their contents in the engineered strain of Saccharomyces cerevisiae by HPLC.

[0108] Shimadzu LC-20AT high performance liquid chromatography, YMC30 column was used for analysis. Column temperature was 40°C, wavelength was 450nm, flow rate was 1mL / min, gradient elution was used, mobile phase was acetonitrile and methanol (75:25) in pump A, and methyl tert-butyl ether was used in pump B. The specific conditions were as follows: 0-10min, mobile phase of pump B increased from 0 to 5%; 10-19min, mobile phase of pump B increased from 5% to 14%; 19-29min, mobile phase of pump B increased from 14 to 25%; 29-54min, mobile phase of pump B increased from 25% to 50%; 54-66min, mobile phase of pump B increased from 50% to 74%; 66.1min, pump B returned to 0.

[0109] HPLC analysis results are as follows Figure 7 As shown, the application of Trx1 or Trx2 solubility tags increased the soluble expression of CYP97A3, a key exogenous enzyme in the lutein synthesis pathway, thereby increasing the production of lutein. Among them, the lutein production of the strain carrying CYP97A3 fused with Trx1 increased by 17.0% compared with the strain without the solubility tag, and the lutein production of the strain carrying CYP97A3 fused with Trx2 increased by 28.6% compared with the strain without the solubility tag.

[0110] Example 5: Application of Trx1 solubilizing expression tag in the synthesis of bisabolene and evaluation of its effect

[0111] (1) Construction of pUMRI vector

[0112] The construction method is the same as in Example 3, thereby constructing pUMRI-T CYC1 -AgBIS-ERG20-Trx1-P GAL1 -P GAL10 -T ADH1 At the same time, a pUMRI vector with SMT3, MBP, and GST tags was constructed as a control.

[0113] (2) Construction of a bisabolene-producing strain carrying a Trx1 solubilizing expression tag

[0114] Using the LiAc / SS carrier DNA / PEG method (High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. Nature Protocols, 2007, 2(1):31-34), T CYC1 -AgBIS-ERG20-Trx1-P GAL1 -P GAL10 -T ADH1 The expression cassette was integrated into the chromosome of BY4742-C04 strain.

[0115] (3) Fermentation culture of bisabolene synthesis and extraction and analysis of products

[0116] The culture conditions are as follows: 50 μL of bacterial solution is taken from the bacterial storage and transferred to a 5 mL YPD test tube, cultured in a shaker at 30°C and 220 rpm for 12-16 hours, then streaked on a YPD plate, grown in a 30°C incubator for 2 days, and then a single colony is picked from the plate and transferred to a 5 mL YPD test tube, cultured in a shaker at 30°C and 220 rpm for 16-24 hours. The initial OD 600The concentration of 0.05 was transferred to a shaking flask containing 50 mL YPD for culture. After 24 hours, dodecane was added for two-phase culture and the culture period was 72 hours to 120 hours.

[0117] The yeast strain producing bisabolene adopts two-phase culture during shake flask fermentation culture, and the specific extraction steps are as follows: 50 mL of fermentation liquid is transferred to a 50 mL centrifuge tube and centrifuged at 3500 rpm for 5 minutes; the fermentation liquid after centrifugation is separated into an upper aqueous phase and a lower oil phase, and the upper oil phase is transferred to a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 1 minute.

[0118] Take 200 μL of the upper oil phase and 50 μL of internal standard dibutyl phthalate and mix them thoroughly; filter the mixed system with a 0.22 μm organic filter into a new 1.5 mL centrifuge tube for GC detection.

[0119] The quantitative analysis of bisabolene uses a gas chromatograph (GC) equipped with a hydrogen flame detector (FID) and an HP-5 capillary column (30m×0.25mm, thickness 0.25μm). When using gas chromatography for detection, 0.1Mpa nitrogen is used as the carrier gas, and the hydrogen is adjusted to 0.1Mpa and the air is adjusted to 0.1Mpa. The injection port temperature is 250℃ and the detector temperature is 320℃. The heating program is shown in the following table.

[0120] Table 2. Heating program

[0121]

[0122] When injecting, use a 1μL injection needle to absorb the sample and eject it all onto a paper towel. Repeat the rinsing of the injection needle 3 to 4 times. Using the scale line of the 1μL injection needle as a standard, quickly inject it into the injection port for gas phase analysis and detection.

[0123] The samples were analyzed by mass spectrometry using a Shimadzu SH-I-5Sil (60m×0.25mm, 0.25μm) column and an Agilent 6890N series gas chromatograph system with a 5973N series mass spectrometer. The injector temperature was 250°C, and the temperature program was: 100°C for 1 min, then heated to 200°C at 40°C / min, then held for 2 min, then heated to 300°C at 40°C / min, then held for 3 min. The carrier gas was helium at a flow rate of 1 mL / min.

[0124] The gas chromatography analysis results are as follows Figure 8 As shown, the Trx1 solubility tag increased the soluble expression of the key exogenous enzyme bisabolene synthase in the bisabolene biosynthesis pathway, thereby increasing the production of bisabolene. The bisabolene production of the strain carrying the bisabolene synthase fused with Trx1 increased by 11.88% compared with the strain without the solubility tag.

[0125] Example 6: Application of Trx1 lysis-promoting expression tag in the synthesis of cantharidin and astaxanthin and its effect evaluation

[0126] (1) Construction of a strain producing cantharidin and astaxanthin carrying a Trx1 lytic expression tag

[0127] The plasmid construction method was the same as in Example 3, thereby constructing pUMRI-T CYC1 -P GAL1 -P GAL10 -Trx1-OBKTM29-T ADH1 and pUMRI-T CYC1 -OcrtZM1-P GAL1 -P GAL10 -Trx1-OBKTM29-T ADH1 .

[0128] At the same time, a pUMRI vector with GST, MBP, NusA, and SMT3 tags was constructed as a control.

[0129] The plasmid digested with Sfil enzyme was chemically transformed into the engineered strain using the LiAc / SS carrier DNA / PEG method (High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. Nature Protocols, 2007, 2(1):31-34). ADH1 -OBKTM29-Trx1-P GAL10 -P GAL1 -T CYC1 or T ADH1 -OBKTM29-Trx1-P GAL10 -P GAL1 -OcrtZM1-T CYC1 The expression cassette was integrated into the chromosome of the β-carotene engineering strain Ycarot-02.

[0130] (2) Fermentation culture of cantharidin and astaxanthin synthesis and extraction and analysis of products

[0131] The fermentation culture and product extraction methods are the same as in Example 4. The analysis method is as follows:

[0132] The following conditions were used to detect various carotenoids and their contents in the engineered strain of Saccharomyces cerevisiae by HPLC. Shimadzu LC-20AT high performance liquid chromatography and Amethyst C18-H column were used for analysis. The column temperature was 40°C, the detection wavelength was 470nm, the flow rate was 1mL / min, and the gradient elution was adopted. The mobile phase was acetonitrile and water (90:10) in pump A and methanol and isopropanol (60:40) in pump B. The specific conditions are as follows: 0-15min, the mobile phase of pump B increased from 0 to 90%, 15-27min, the mobile phase of pump B was maintained at 90%, 27-28min, the mobile phase of pump B was reduced from 90% to 0, and 28-35min, the mobile phase of pump B was maintained at 0.

[0133] HPLC analysis results showed that the Trx1 solubility-promoting tag promoted the soluble expression of β-carotene ketolase, a key exogenous enzyme in the biosynthesis pathway of Cantharides, thereby increasing the yield of Cantharides. The Cantharides yield of the strain carrying β-carotene ketolase fused with Trx1 increased by 16.2% ( Fig. 9 ). Similarly, as a downstream product of canthaxanthin, in the astaxanthin synthesis pathway, the Trx1 solubility-promoting tag increases the accumulation of β-carotene ketosylation products canthaxanthin and echinone by promoting the soluble expression of β-carotene ketoylase, thereby increasing the production of its downstream product astaxanthin by 12.4% and the ketosylation carotenoid product by 18.6% ( Fig.10 ).

[0134] The above experimental results prove that the Trx1 or Trx2 soluble expression tag derived from Saccharomyces cerevisiae can help the exogenous fusion protein to fold correctly in Saccharomyces cerevisiae, increase the soluble expression level of the exogenous protein in Saccharomyces cerevisiae, and effectively promote the heterologous biosynthesis of the target natural product.

Claims

1. A use of yeast-derived thioredoxin as a solubility-promoting expression tag, characterized in that: The amino acid sequence of the thioredoxin is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The lytic expression tag is a lytic expression tag of a yeast expression system.

3. The use according to claim 1 or 2, characterized in that: The application includes: inserting the coding sequence of the soluble expression tag into the upstream of the foreign protein coding gene in the gene expression box, and performing fusion expression with the foreign protein coding gene in the host cell to improve the soluble expression of the foreign protein.

4. The use according to claim 3, characterized in that The gene expression box includes a promoter, a foreign protein encoding gene and a terminator, and the coding sequence of the lytic expression tag is inserted downstream of the promoter.

5. The use according to claim 3, characterized in that The host cell is Saccharomyces cerevisiae.

6. The use according to claim 3, characterized in that The coding sequence of the lytic expression tag is shown as SEQ ID NO.3 or SEQ ID NO.

4.

7. The use according to claim 3, characterized in that The application includes: using yeast as a starting bacterium, integrating a gene expression box inserted with a soluble expression tag coding sequence into a yeast chromosome through an integration plasmid, and obtaining a genetically engineered bacterium that solublely expresses foreign proteins; Alternatively, yeast is used as a starting bacterium, and a recombinant expression plasmid containing a gene expression cassette inserted with a lytic expression tag coding sequence is introduced to obtain a genetically engineered bacterium that solublely expresses foreign proteins.

8. The use according to claim 7, characterized in that The integration plasmid is a pUMRI series assembly tool plasmid.

9. The use according to claim 7, characterized in that The genetically engineered bacteria are genetically engineered bacteria that produce lutein, bisabolene, canthaxanthin or astaxanthin.

10. The use according to claim 9, characterized in that The exogenous proteins are carotene β-hydroxylase from Arabidopsis thaliana, bisabolene synthase from Abies grandis, and β-carotene ketolase from Haematococcus pluvialis.

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