Recombinant Yarrowia lipolytica engineering strain for producing taxadiene and application of recombinant Yarrowia lipolytica engineering strain

Abstract

The invention discloses a recombinant Yarrowia lipolytica engineering strain for producing taxadiene and application of the recombinant Yarrowia lipolytica engineering strain, and belongs to the technical field of genetic engineering and bioengineering. According to the present invention, the recombinant Yarrowia lipolytica capable of synthesizing the taxadiene is obtained by expressing the recombinant Yarrowia lipolytica of the taxadiene synthase TXS, the endogenous MVA biosynthesis pathway is further strengthened, the squalene pathway is weakened, the GGPP supply is improved, the heteroditerpene synthase and the geranyl-geranyl pyrophosphate synthase are constructed and strengthened, and the TXS can be used for synthesizing the taxadiene; according to the method, the yield of the taxadiene is increased, the yield of the taxadiene of the Yarrowia lipolytica fermented for 120 hours is finally increased by 262.1 times, the final shake flask fermentation yield is 58.7 + / -3.8 mg / L, and a foundation is laid for subsequent paclitaxel biosynthesis.

Description

Technical Field

[0001] The present invention relates to a recombinant Yarrowia lipolytica engineering strain for producing taxadiene and its application, belonging to the technical fields of genetic engineering and bioengineering. Background Art

[0002] Taxadiene is the first cyclized product in the biosynthetic pathway of paclitaxel and is also a key intermediate in the synthesis of all taxane natural products. Paclitaxel is a diterpenoid compound with significant anti-cancer activity and is widely used in the treatment of various types of solid cancers, such as ovarian cancer, breast cancer, lung cancer, and gastric cancer, etc. Paclitaxel has significant effects and low side effects in the treatment of the above cancers. Due to its unique anti-cancer mechanism, broad-spectrum anti-tumor activity, significant clinical efficacy, and important position in the treatment of various cancers, paclitaxel is known as the "star anti-cancer molecule". The global market has a huge demand for paclitaxel. The market value of global paclitaxel injection in 2024 was 4.17 billion US dollars, of which the market scale in China reached 1.371 billion US dollars. It is expected that the global market value of paclitaxel will reach 8.626 billion US dollars in 2031 at an annual growth rate of 11.1%.

[0003] However, paclitaxel mainly comes from extracting the bark of endangered plants Taxus chinensis, with low yield and high extraction cost, which severely restricts its wide application. In addition, chemical synthesis is considered a non-green and sustainable production method due to factors such as reagent toxicity and product yield. Therefore, it is crucial to develop a sustainable production method for paclitaxel. Microbial fermentation to produce paclitaxel or its precursors is considered a highly potential alternative. As the first key precursor in the paclitaxel synthesis pathway, the increase in the yield of taxadiene is of great significance for the biosynthesis of paclitaxel. Therefore, aiming at the limitations in aspects such as the extraction of natural products and the chemical synthesis of paclitaxel, product quality safety, and price advantages, using synthetic biology technology to construct a new cell factory to produce taxadiene in large quantities lays a solid foundation for the synthesis of paclitaxel. Summary of the Invention

[0004] The present invention provides a Yarrowia lipolytica engineering bacterium producing taxadiene, which expresses the taxadiene synthase gene TXS derived from Taxus canadensis.

[0005] In one embodiment, the nucleotide sequence of the taxadiene synthase gene TXS is as shown in SEQ ID NO.1.

[0006] In one embodiment, the Yarrowia lipolytica engineering bacterium further has at least one of the following improvements:

[0007] (1) Strengthen the expression of endogenous MVA biosynthetic pathway genes;

[0008] (2) Express the multifunctional diterpene synthase gene tPaGGPPs derived from Diaporthe amygdali and the geranylgeranyl pyrophosphate synthase gene XdGGPPs derived from Phaffia rhodozyma;

[0009] (3) Use promoter P ERG1 to replace promoter P SQS1 .

[0010] In one embodiment, the endogenous MVA biosynthetic pathway genes include tHMG1, ERG12, ERG20, and IDI1.

[0011] In one embodiment, the starting strain is Yarrowia lipolytica with Ku70 knocked out.

[0012] In one embodiment, the engineered Yarrowia lipolytica strain integrates the gene TXS at the IntE-3 locus based on Yarrowia lipolytica Ku70.

[0013] In one embodiment, the engineered Yarrowia lipolytica strain integrates the gene TXS at the IntE-3 locus based on Yarrowia lipolytica Ku70, overexpresses the endogenous genes tHMG1, ERG12, ERG20, and IDI1 of Yarrowia lipolytica.

[0014] In one embodiment, the engineered Yarrowia lipolytica strain integrates the gene TXS at the IntE-3 locus based on Yarrowia lipolytica Ku70, overexpresses the endogenous genes tHMG1, ERG12, ERG20, and IDI1 of Yarrowia lipolytica, introduces the multifunctional diterpene synthase gene tPaGGPPs derived from Diaporthe amygdali, and overexpresses the geranylgeranyl pyrophosphate synthase gene XdGGPPs of Phaffia rhodozyma.

[0015] In one embodiment, the engineered Yarrowia lipolytica strain integrates the gene TXS at the IntE-3 locus based on Yarrowia lipolytica Ku70, overexpresses the endogenous genes tHMG1, ERG12, ERG20, and IDI1 of Yarrowia lipolytica, introduces the multifunctional diterpene synthase gene tPaGGPPs of Diaporthe amygdali, and overexpresses the geranylgeranyl pyrophosphate synthase gene XdGGPPs of Phaffia rhodozyma, and uses promoter P ERG1 to replace the original promoter of the SQS1 gene.

[0016] In one embodiment, the nucleotide sequence of the SQS1 gene is as shown in GenBank: AF092497.1.

[0017] In one embodiment, utilize promoter PTEFin Initiate TXS gene expression.

[0018] In one embodiment, the nucleotide sequence of the gene tHMG1 is as shown in SEQ ID NO.2; the nucleotide sequence of the gene ERG12 is as shown in SEQ ID NO.3; the nucleotide sequence of the gene ERG20 is as shown in SEQ ID NO.4; the nucleotide sequence of the gene IDI1 is as shown in SEQ ID NO.5.

[0019] In one embodiment, the MVA biosynthetic pathway genes are integrated at the Trp (GenBank: AF420590.1) locus.

[0020] In one embodiment, use promoter P TEF Initiate the expression of the tHMG1 gene, ERG12 gene, ERG20 gene, and IDI1 gene.

[0021] In one embodiment, the nucleotide sequence of the multifunctional diterpene synthase gene tPaGGPPs is as shown in SEQ ID NO.6; the nucleotide sequence of the geranylgeranyl pyrophosphate synthase gene XdGGPPs is as shown in SEQ ID NO.7.

[0022] In one embodiment, use promoter P TEF Initiate the expression of the XdGGPPs gene, and use promoter P TDH Initiate tPaGGPPs.

[0023] In one embodiment, the Yarrowia lipolytica is Yarrowia lipolytica Ku70, which has been published in the paper "De Novo Biosynthesis of Lutein in Yarrowia lipolytica".

[0024] The present invention also provides the application of the engineered Yarrowia lipolytica in the production of taxadiene.

[0025] In one embodiment, the engineered Yarrowia lipolytica is fermented in a medium at 28 - 30 °C for at least 72 h.

[0026] In one embodiment, the engineered Yarrowia lipolytica is fermented in a medium at 28 - 30 °C for 72 - 120 h.

[0027] In one embodiment, the medium includes but is not limited to YPD medium.

[0028] The present invention also claims the application of the engineered Yarrowia lipolytica in the production of products containing taxadiene in the fields of food, medicine, and chemical industry.

[0029] In one embodiment, the application is for preparing a drug with taxadiene as a precursor.

[0030] In one embodiment, the drug includes but is not limited to paclitaxel.

[0031] Beneficial effects: In the present invention, Yarrowia lipolytica Ku70 is used as a host, and recombinant Yarrowia lipolytica expressing the taxadiene synthase gene TXS is used to obtain recombinant Yarrowia lipolytica capable of synthesizing taxadiene. Further, the endogenous MVA biosynthetic pathway is strengthened, the squalene synthesis pathway is weakened, the supply of GGPP is increased, the heterologous diterpene synthase gene and geranylgeranyl pyrophosphate synthase gene are constructed and strengthened, the yield of taxadiene is increased, and the taxadiene yield of Yarrowia lipolytica after 120 h of shake flask fermentation is increased by 262.1 times, reaching 58.7 ± 3.8 mg / L, reaching the highest level of shake flask fermentation yield in current Yarrowia lipolytica, laying a foundation for subsequent paclitaxel biosynthesis. Description of the Drawings

[0032] Figure 1 It is a metabolic schematic diagram of heterologous synthesis of taxadiene in Yarrowia lipolytica; wherein HMG-CoA, 3-hydroxy-3-methyl-glutaryl coenzyme A; MVA, mevalonic acid; MVAP, 5-phosphomevalonic acid; MVAPP, mevalonic acid; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; green arrows represent strengthening, blue arrows represent weakening, and dotted lines represent multiple-step reactions.

[0033] Figure 2 It is the GC-MS detection result of taxadiene after the engineered strain is cultured in YPD; wherein, a, the ion chromatogram of taxadiene; b, the ion mass spectrum of taxadiene; c, the ion current intensity of taxadiene in the fermentation broth of strain Tax1.

[0034] Figure 3 It is the taxadiene content in the fermentation broth of different strains in shake flask fermentation. Detailed Embodiments

[0035] (I) Culture Medium

[0036] LB culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride. Add 20 g / L agar powder to prepare LB solid culture medium.

[0037] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L. For auxotrophic media, corresponding amino acids need to be added, and the specific addition amounts are: uracil 0.05 g / L, tryptophan 0.05 g / L, leucine 0.05 g / L.

[0038] YPD medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L.

[0039] (2) Preparation of competent cells of Yarrowia lipolytica: The Frozen-EZ Yeast Transformation II transformation kit is used for the preparation of competent cells of Yarrowia lipolytica. Take 2 mL of YPD medium to culture Yarrowia lipolytica (OD 600 = 0.8 - 1.0) and perform the following operation steps.

[0040] 1. Centrifuge the cells at 3500 rpm for 5 min, and aspirate the supernatant.

[0041] 2. Add 500 μL of Buffer1 solution to wash the pellet, centrifuge the pellet cells again, and aspirate the supernatant.

[0042] 3. Resuspend the pellet cells with 50 μL of Buffer 2 solution.

[0043] (3) Transformation of Yarrowia lipolytica:

[0044] 1. Mix the 50 μL of competent cells prepared above with 5 μg of linear DNA fragment and 1 μg of CRISPR plasmid; add 500 μL of Buffer3 solution and mix well.

[0045] 2. Incubate at 30 °C for 60 min, and gently flick with fingers or vortex at low speed 2 - 3 times during the incubation to mix well.

[0046] 3. Centrifuge the cells at 5000 rpm for 5 min, discard the supernatant, add 1 mL of YPD medium and incubate at 30 °C for 2 h.

[0047] 4. Centrifuge the incubation solution at 5000 rpm for 5 min, discard the supernatant, resuspend the cell pellet with 100 μL of sterile PBS and spread it on an appropriate auxotrophic plate.

[0048] 5. Incubate the plate at 30 °C for 3 days to grow transformants.

[0049] (4) Determination of taxadiene by GC-MS: The determination was carried out using a Shimadzu gas chromatography-mass spectrometry tandem instrument. GC-MS conditions: The inlet temperature was set at 280 °C, in split mode, with a split ratio of 10:1. The injection volume was 1 μl. The initial temperature was 130 °C and was held for 2 min, then heated at a rate of 8 °C / min to 250 °C, and then heated at a rate of 10 °C / min to 310 °C and held at 310 °C for 5 min. Then it was rapidly heated to 320 °C and held for 3 min. After a 6-min solvent delay, MS data in the mass range of 50 - 550 m / z were collected.

[0050] (5) The strain information is shown in Table 1.

[0051] Table 1 Strains involved in the present invention

[0052]

[0053] (6) The promoters / terminators involved in the examples are shown in Table 2, and the primer sequences are shown in Table 3.

[0054] Table 2 Promoters / terminators involved in the examples

[0055]

[0056]

[0057]

[0058] Table 3 Primer sequences

[0059]

[0060]

[0061] Example 1: Construction of a recombinant Yarrowia lipolytica strain containing taxadiene synthase

[0062] The IntE-3 site of Yarrowia lipolytica with Ku70 knocked out was selected as the integration site for taxadiene synthase TXS (nucleotide sequence as shown in SEQ ID NO.1). The specific steps are as follows:

[0063] The TXS gene shown in SEQ ID NO.1 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The promoter P was amplified from the Yarrowia lipolytica Po1f genome using the primers F1 / R1 in Table 3 TEFin , and the terminator T was amplified using the primers F2 / R2 XPR, the upstream homologous arm 523bp sequence and the downstream homologous arm 469bp sequence of the IntE-3 locus (shown in SEQ ID NO.8) were amplified using primers F3 / R3 and F4 / R4 respectively. The above fragments were assembled by OE-PCR. The PCR products were recovered by ethanol precipitation method. Approximately 5μg of the integration fragment and approximately 1μg of sgRNA were transformed into the Yarrowia lipolytica strain Ku70 using the Yeast Transformation Kit Frozen-EZ Yeast TransformationII, spread on the screening solid medium, and cultured at 30°C for 3 days until colonies appeared. The correct clones were picked and named Saccharomyces cerevisiae Tax1.

[0064] The obtained engineered strain Tax1 was cultured in YPD medium, 10% dodecane was added, and fermentation was carried out at 30°C and 220rpm for 120h. The content of the product was detected by GC-MS. The results showed that taxadiene was contained in the dodecane of the organic phase layer of the fermentation broth of Yarrowia lipolytica Tax1, and the yield was 0.224mg / L.

[0065] Example 2: Optimization of the endogenous MVA biosynthetic pathway

[0066] To promote the supply of GGPP, the endogenous genes tHMG1 (nucleotide sequence as SEQ ID NO.2), ERG12 (nucleotide sequence as SEQ ID NO.3), ERG20 (nucleotide sequence as SEQ ID NO.4), and IDI1 (nucleotide sequence as SEQ ID NO.5) of Yarrowia lipolytica were overexpressed. After the above genes were concatenated, they were respectively integrated into Trp (GenBank: AF420590.1) and the IntE-4 locus (shown in SEQ ID NO.9). The specific steps were as follows:

[0067] Primers F5 / R5, F6 / R6, F7 / R7, and F8 / R8 were used to amplify the genes tHMG1, ERG12, ERG20, and IDI1 from the Yarrowia lipolytica Po1f genome respectively. Primer F9 / R9 in Table 3 was used to amplify the promoter P TEF , primer F10 / R10 was used to amplify the terminator T LIP , primers F11 / R11 and F12 / R12 were used to amplify the upstream homologous arm 1075bp sequence and the downstream homologous arm 1087bp sequence of the Trp locus from the Yarrowia lipolytica Po1f genome respectively. Primers F13 / R13 and F14 / R14 were used to amplify the upstream homologous arm 520bp sequence and the downstream homologous arm 475bp sequence of the IntE-4 locus from the Yarrowia lipolytica Po1f genome respectively. The above fragments were assembled by OE-PCR. The PCR products were recovered by ethanol precipitation method. Approximately 5μg of the integration fragment (P TEFin-URA3-T XPR -P TEF -tHMG1-T LIP -P TEF -ERG12-T LIP -P TEF -ERG20-T LIP -P TEF -IDI1-T LIP ) Approximately 1 μg of sgRNA was transformed into the engineered strain Tax1 of Yarrowia lipolytica using the Yeast Transformation Kit Frozen-EZ Yeast Transformation II, spread on a selective solid medium, and cultured at 30 °C for 3 days until colonies appeared. The correct clones were picked and named Yarrowia lipolytica Tax2 (integrating the above genes at the Trp locus) and Tax3 (integrating the above genes at the IntE-4 locus). The obtained engineered strains Tax2 and Tax3 were cultured in YPD medium supplemented with 10% dodecane and fermented at 30 °C and 220 rpm for 120 h. The content of the product was detected by GC-MS. The results showed that the taxadiene yields of the strains constructed by integrating genes at different loci varied greatly. Among them, the taxadiene yield of strain Tax2 reached 1.651 mg / L, which was 5.2 times higher than that of strain Tax3 (0.318 mg / L).

[0068] Example 3: Expression of heterologous multifunctional diterpene synthase and geranylgeranyl pyrophosphate synthase to enhance taxadiene synthesis

[0069] The tPaGGPPs gene shown in SEQ ID NO.6 and the XdGGPPs gene shown in SEQ ID NO.7 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The promoter P was amplified from the genome of Yarrowia lipolytica Po1f using primers F15 / R15 TEF , and the terminator T was amplified from the genome of Yarrowia lipolytica Po1f using primers F16 / R16 XPR , the promoter P was amplified from the genome of Yarrowia lipolytica Po1f using primers F17 / R17 TDH , and the terminator T was amplified from the genome of Yarrowia lipolytica Po1f using primers F18 / R18 ICL, the upstream homologous arm 500bp sequence and downstream homologous arm 467bp sequence of the IntE-1 locus (shown in SEQ ID NO.10) were amplified from the Yarrowia lipolytica Po1f genome using primers F19 / R19 and F20 / R0. The upstream homologous arm 985bp sequence and downstream homologous arm 997bp sequence of the AXP locus (shown in SEQ ID NO.11) were amplified from the Yarrowia lipolytica Po1f genome using primers F21 / R21 and F22 / R22. The above fragments were assembled by OE-PCR. The PCR products were recovered by ethanol precipitation. Approximately 5μg of the integration fragment (P TEF -XdGGPP-T XPR -P TDH -tPaGGPP-T ICL ) and approximately 1μg of sgRNA were transformed into the Yarrowia lipolytica engineering strain Tax2 using the Yeast Transformation Kit Frozen-EZ Yeast Transformation II, spread on the screening solid medium, and cultured at 30°C for 3 days until colonies appeared. The correctly verified clones were picked and named Yarrowia lipolytica Tax4 (integrating the above gene at the IntE-1 locus) and Tax5 (integrating the above gene at the AXP locus). The obtained engineering strains Tax4 and Tax5 were cultured in YPD medium respectively, 10% dodecane was added, and fermentation was carried out at 30°C and 220rpm for 120h. The content of the product was detected by GC-MS. The results showed that the taxadiene yield of the strain Tax4 with the gene integrated at the IntE-1 locus reached 48.255mg / L, and the taxadiene yield of the strain Tax5 with the gene integrated at the AXP locus was 34.468mg / L.

[0070] Example 4: Weakening of squalene

[0071] To further promote the synthesis of taxadiene, the metabolic flux of the squalene pathway was weakened, thereby enhancing the carbon flux of the GGPP pathway. The promoter P SQS1 regulating the transcription of squalene synthase was replaced with the endogenous weak promoter P ERG1 of Yarrowia lipolytica.

[0072] The specific steps are as follows:

[0073] The weak promoter P ERG1 was amplified from the Yarrowia lipolytica Po1f genome using primers F23 / R23, and the original promoter P SQS1The upstream homologous arm 1092bp sequence and the downstream homologous arm 1025bp sequence. The above fragments were assembled using OE-PCR. The PCR products were recovered by ethanol precipitation. Approximately 5 μg of the integration fragment and approximately 1 μg of sgRNA were transformed into the engineered strain Tax4 of Yarrowia lipolytica using the Frozen-EZ Yeast Transformation II kit, spread on the screening solid medium, and cultured at 30 °C for 3 days until colonies appeared. The correctly verified clones were picked and named Yarrowia lipolytica Tax6. The obtained engineered strain Tax6 was cultured in YPD medium, 10% dodecane was added, and fermentation was carried out at 30 °C and 220 rpm for 120 h. GC-MS was used to detect the content of the product. The results showed that the weakening of squalene helped to increase the yield of taxadiene. The original promoter P SQS1 was replaced with P ERG1 . After that, the taxadiene yield of the constructed strain Tax6 was 58.7 ± 3.8 mg / L, which was the strain with the highest taxadiene yield in shake flask fermentation in Yarrowia lipolytica at present.

[0074] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An engineered strain of Yarrowia lipolytica producing taxadiene, characterized in that: The taxadiene synthase encoding gene TXS from Taxus canadensis was expressed; the nucleotide sequence of the gene TXS is shown in SEQ ID NO.

1.

2. The engineered yeast Yarrowia lipolytica according to claim 1, characterized in that There is also at least one of the following improvements: (1) Enhance the expression of endogenous MVA biosynthetic pathway genes; (2) expressing the multifunctional diterpene synthase gene tPaGGPPs from Diaporthe amygdali and the geranylgeranyl pyrophosphate synthase gene XdGGPPs from Phaffia rhodozyma; (3) Using promoter P ERG1 Regulates the expression of the SQS1 gene.

3. The engineered yeast Yarrowia lipolytica according to claim 2, characterized in that The endogenous MVA biosynthetic pathway genes include tHMG1, ERG12, ERG20 and IDI1.

4. The engineered yeast Yarrowia lipolytica according to any one of claims 1 to 3, characterized in that Using promoter P TEFin Initiation of TXS gene expression.

5. The engineered yeast Yarrowia lipolytica according to any one of claims 2 to 4, characterized in that The MVA biosynthetic pathway genes were integrated at the Trp locus.

6. The engineered yeast Yarrowia lipolytica according to claim 5, characterized in that With promoter P TEF Initiate the expression of tHMG1, ERG12, ERG20 and IDI1 genes.

7. The engineered yeast Yarrowia lipolytica according to any one of claims 2 to 6, characterized in that With promoter P TEF Initiate the expression of XdGGPPs gene and use the promoter P TDH Initiate expression of tPaGGPPs gene.

8. The engineered yeast Yarrowia lipolytica according to any one of claims 1 to 7, characterized in that The starting strain was Yarrowia lipolytica in which Ku70 was knocked out.

9. A method for preparing taxadiene, characterized in that: The engineered Yarrowia lipolytica according to any one of claims 1 to 8 is cultured in a culture medium, and the taxadiene in the fermentation broth is collected.

10. The method according to claim 9, characterized in that The engineered Yarrowia lipolytica is fermented in a culture medium at 28-30° C. for at least 72 hours.

11. Use of the engineered Yarrowia lipolytica according to any one of claims 1 to 8 in producing products containing taxadiene in the fields of food, medicine or chemical industry.

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