Recombinant yarrowia lipolytica engineering strain for producing taxadiene and application thereof

Abstract

The application discloses a recombinant L lipomyces starkeyi engineering strain for producing taxadiene and application, and belongs to the technical field of genetic engineering and bioengineering.The recombinant L lipomyces starkeyi for expressing taxadiene synthase TXS is used to obtain the recombinant L lipomyces starkeyi capable of synthesizing taxadiene, the endogenous MVA biosynthesis pathway is further strengthened, the squalene pathway is weakened, the supply amount of GGPP is increased, the heterologous diterpene synthase and the geranylgeranyl pyrophosphate synthase are constructed and strengthened, and the yield of taxadiene is increased, so that the yield of taxadiene of the L lipomyces starkeyi after 120h of fermentation is finally increased by 262.1 times, and the final shake flask fermentation yield is 58.7+ / -3.8mg / L, which lays a foundation for subsequent paclitaxel biosynthesis.

Description

Technical Field

[0001] This invention relates to a recombinant lipophilic yeast strain for producing taxadiene and its applications, belonging to the fields of genetic engineering and bioengineering technology. Background Technology

[0002] Taxadiene is the first cyclization product in the biosynthetic pathway of paclitaxel and a key intermediate in the synthesis of all taxane natural products. Paclitaxel is a diterpenoid compound with significant anticancer activity and is widely used to treat various types of solid cancers, such as ovarian cancer, breast cancer, lung cancer, and gastric cancer. Paclitaxel is highly effective in treating these cancers with low side effects. Due to its unique anticancer mechanism, broad-spectrum antitumor activity, significant clinical efficacy, and important role in the treatment of various cancers, paclitaxel is hailed as a "star anticancer molecule." The global market demand for paclitaxel is enormous. In 2024, the global market value of paclitaxel injections reached US$4.17 billion, of which the Chinese market size reached US$1.371 billion. The global market value of paclitaxel is projected to reach US$8.626 billion by 2031, with an annual growth rate of 11.1%.

[0003] However, paclitaxel is mainly derived from the bark of the endangered yew tree, resulting in low yields and high extraction costs, severely limiting its widespread application. Furthermore, chemical synthesis is considered a non-green and unsustainable production method due to factors such as reagent toxicity and product yield. Therefore, developing sustainable paclitaxel production methods is crucial. Microbial fermentation for the production of paclitaxel or its precursors is considered a highly promising alternative. Taxadiene, as the first key precursor in the paclitaxel synthesis pathway, is of great significance for improving its biosynthesis. Therefore, addressing the limitations of natural product extraction and chemical synthesis of paclitaxel in terms of product quality, safety, and price advantages, utilizing synthetic biology techniques to construct novel cell factories for the large-scale production of taxadiene lays a solid foundation for paclitaxel synthesis. Summary of the Invention

[0004] This invention provides a taxadiene-producing engineered yeast strain, which expresses the taxadiene synthase gene TXS derived from Taxusscanadensis.

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

[0006] In one embodiment, the engineered *Yamylostella lipolytica* strain further has at least one of the following improvements:

[0007] (1) Enhance the expression of genes involved in the endogenous MVA biosynthesis pathway;

[0008] (2) Expression of the multifunctional diterpenoid synthase gene tPaGGPPs derived from Diaporthe amygdali and the geranylgeranyl pyrophosphate synthase gene XdGGPPs derived from Rhodotorula rubra.

[0009] (3) Using promoter P ERG1 Replace the promoter P SQS1 .

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

[0011] In one implementation, the starting strain is a Ku70-knockout *Yarrowia lipophila*.

[0012] In one embodiment, the engineered *Yolopsis lipolytica* strain is based on *Yolopsis lipolytica* Ku70, with the TXS gene integrated at the IntE-3 site.

[0013] In one embodiment, the engineered *Yolopsis lipolytica* strain is based on *Yolopsis lipolytica* Ku70, with the gene TXS integrated at the IntE-3 site and overexpression of the endogenous genes tHMG1, ERG12, ERG20, and IDI1.

[0014] In one embodiment, the engineered *Yamyces lipolytica* strain is based on *Yamyces lipolytica* Ku70, with the TXS gene integrated at the IntE-3 site and overexpression of the endogenous genes tHMG1, ERG12, ERG20, and IDI1. It also incorporates the multifunctional diterpenoid synthase gene tPaGGPPs from *Diaporthe amygdali* and overexpresses the geranylgeranyl pyrophosphate synthase gene XdGGPPs from *Rhodotorula rubra*.

[0015] In one embodiment, the engineered *Yamyces lipolytica* strain is based on *Yamyces lipolytica* Ku70, with the TXS gene integrated at the IntE-3 site and overexpression of the endogenous genes tHMG1, ERG12, ERG20, and IDI1. It also incorporates the multifunctional diterpenoid synthase gene tPaGGPPs from *Diaporthe amygdali* and overexpresses the geranylgeranyl pyrophosphate synthase gene XdGGPPs from *Rhodotorula rubiginosa*, and uses the promoter P... ERG1 Replace the original promoter of the SQS1 gene.

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

[0017] In one implementation, promoter P is used.TEFin Initiate TXS gene expression.

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

[0019] In one embodiment, the MVA biosynthetic pathway gene is integrated at the Trp (GenBank: AF420590.1) site.

[0020] In one implementation, promoter P is used. TEF Expression of the initiating tHMG1, ERG12, ERG20 and IDI1 genes.

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

[0022] In one implementation, promoter P is used. TEF Initiating XdGGPPs gene expression using promoter P TDH Start tPaGGPPs.

[0023] In one embodiment, the *Yarrowia lipolytica* is *Yarrowia lipolytica* Ku70, which has been disclosed in the paper "DeNovo Biosynthesis of Lutein in Yarrowia lipolytica".

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

[0025] In one embodiment, the engineered *Yamylostella lipolytica* strain is fermented in a culture medium at 28–30°C for at least 72 hours.

[0026] In one embodiment, the engineered *Yamylostella lipolytica* strain is fermented in a culture medium at 28–30°C for 72–120 h.

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

[0028] This invention also claims protection for the use of the engineered *Yamylostella lipolytica* strain in the production of taxadiene-containing products in the food, pharmaceutical, and chemical industries.

[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: This invention uses *Yamylostella lipolytica* Ku70 as the host and obtains a recombinant *Yamylostella lipolytica* capable of synthesizing taxadiene by expressing the taxadiene synthase gene TXS. Furthermore, the endogenous MVA biosynthesis pathway is enhanced, the squalene synthesis pathway is weakened, and the supply of GGPP is increased. The heteroditerpene synthase gene and gerany-gerany-pyrophosphate synthase gene are constructed and enhanced, increasing the taxadiene yield. The taxadiene yield of *Yamylostella lipolytica* after 120 hours of shake-flask fermentation is increased by 262.1 times to 58.7 ± 3.8 mg / L, reaching the highest yield level of shake-flask fermentation in *Yamylostella lipolytica* to date, laying the foundation for subsequent paclitaxel biosynthesis. Attached Figure Description

[0032] Figure 1 This is a metabolic diagram illustrating the heterologous synthesis of taxadiene in *Yamylostella lipolytica*; where HMG-CoA is 3-hydroxy-3-methylglutaryl-CoA; MVA is mevalonate; MVAP is mevalonate-5-phosphate; MVAPP is glutarate; IPP isoprenyl pyrophosphate; DMAPP is dimethylallyl pyrophosphate; FPP is farnesyl pyrophosphate; and GGPP is geraniylgeraniyl pyrophosphate. Green arrows represent enhancement, blue arrows represent weakening, and dashed lines represent multi-step reactions.

[0033] Figure 2 The results of GC-MS detection of taxadiene in the engineered strain after YPD culture are shown; where a, taxadiene ion current chromatogram; b, taxadiene ion current mass spectrum; c, taxadiene ion current intensity in the fermentation broth of strain Tax1.

[0034] Figure 3 The content of taxadiene in the fermentation broth of different strains during shake-flask fermentation. Detailed Implementation

[0035] (I) Culture medium

[0036] LB 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 medium.

[0037] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L. For auxotrophic media, the corresponding amino acids need to be added, specifically: 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] (II) Preparation of competent cells of *Yamylostella lipolytica*: Competent cells of *Yamylostella lipolytica* were prepared using the Frozen-EZ Yeast Transformation II kit. 2 mL of YPD medium was used to culture *Yamylostella lipolytica* (OD2000). 600 (=0.8-1.0) Perform the following operation steps.

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

[0041] 2. Add 500 μL of Buffer 1 solution to wash the precipitate, centrifuge the precipitate cells again, and aspirate the supernatant;

[0042] 3. Add 50 μL of Buffer 2 solution to resuspend the precipitated cells.

[0043] (III) Transformation of Yeast Extract:

[0044] 1. Mix 50 μL of competent cells prepared above with 5 μg of linear DNA fragment and 1 μg of CRISPR plasmid; add 500 μL of Buffer 3 solution and mix thoroughly;

[0045] 2. Incubate at 30℃ for 60 minutes. During incubation, gently tap or vortex the mixture 2-3 times with your fingers to mix it thoroughly.

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

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

[0048] 5. Transformants were grown after incubation on a plate at 30℃ for 3 days.

[0049] (IV) Taxadiene GC-MS Determination: Determination was performed using a Shimadzu gas chromatography-mass spectrometer in tandem. GC-MS conditions: Injector temperature set to 280℃, split mode, split ratio 10:1. Injection volume: 1 μl. Initial temperature: 130℃, held for 2 min. Temperature ramped to 250℃ at 8℃ / min, then to 310℃ at 10℃ / min and held for 5 min. The temperature was then rapidly ramped to 320℃ and held for 3 min. After a 6 min solvent delay, MS data were collected in the mass range of 50–550 m / z.

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

[0051] Table 1. Strains involved in this invention

[0052]

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

[0054] Table 2 shows the promoters / terminators involved in the embodiments.

[0055]

[0056]

[0057]

[0058] Table 3 Primer sequences

[0059]

[0060]

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

[0062] The IntE-3 site of *Yamanthus lipolyticus* 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., and the promoter P was amplified from the Po1f genome of *Agropyron lipolytica* using primers F1 / R1 in Table 3. TEFin The terminator T was amplified using primers F2 / R2. XPRThe upstream homologous arm (523 bp) and downstream homologous arm (469 bp) of the IntE-3 site (SEQ ID NO. 8) were amplified using primers F3 / R3 and F4 / R4, respectively. The fragment was assembled using OE-PCR. The PCR product was recovered by ethanol precipitation. Approximately 5 μg of the integrated fragment and approximately 1 μg of sgRNA were transformed into *Saccharomyces lipolyticus* strain Ku70 using the Frozen-EZ Yeast Transformation II kit. The transformed samples were plated on selection solid medium and incubated at 30°C for 3 days until colonies appeared. The correct clone was selected and named *Saccharomyces cerevisiae* Tax1.

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

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

[0066] To promote GGPP supply, the endogenous genes tHMG1 (nucleotide sequence as shown in SEQ ID NO. 2), ERG12 (nucleotide sequence as shown in SEQ ID NO. 3), ERG20 (nucleotide sequence as shown in SEQ ID NO. 4), and IDI1 (nucleotide sequence as shown in SEQ ID NO. 5) of *Yamaceae lipolytica* were overexpressed. These genes were then tandemly integrated into the Trp (GenBank: AF420590.1) and IntE-4 sites (as shown in SEQ ID NO. 9), respectively. The specific steps are as follows:

[0067] Genes tHMG1, ERG12, ERG20, and IDI1 were amplified from the *Yamyces lipolytica* Po1f genome using primers F5 / R5, F6 / R6, F7 / R7, and F8 / R8, respectively. Promoter P was amplified from the *Yamyces lipolytica* Po1f genome using primer F9 / R9 (listed in Table 3). TEF The terminator T was amplified using primers F10 / R10. LIP The 1075 bp upstream and 1087 bp downstream homologous arms of the Trp site were amplified from the *Agropyron lipolytica* Po1f genome using primers F11 / R11 and F12 / R12, respectively. The 520 bp upstream and 475 bp downstream homologous arms of the IntE-4 site were amplified from the *Agropyron lipolytica* Po1f genome using primers F13 / R13 and F14 / R14, respectively. The fragments were then assembled using OE-PCR. The PCR products were recovered by ethanol precipitation. Approximately 5 μg of the integrated fragment (P...) was obtained. 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 *Yacinthia lipolytica* strain Tax1 using the Frozen-EZ Yeast Transformation II kit. The transformed strain was plated on screening solid medium and incubated at 30°C for 3 days until colonies appeared. Correct clones were selected and named *Yacinthia lipolytica* Tax2 (integrating the above gene at the Trp site) and Tax3 (integrating the above gene at the IntE-4 site), respectively. The resulting engineered strains Tax2 and Tax3 were cultured in YPD medium with 10% dodecane added and fermented at 30°C and 220 rpm for 120 h. The product content was detected by GC-MS. The results showed that the taxadiene yield of strains with genes integrated at different sites varied significantly. Strain Tax2 achieved a taxadiene yield of 1.651 mg / L, which was 5.2 times higher than the 0.318 mg / L yield of strain Tax3.

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

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

[0070] Example 4: Weakening of squalene

[0071] To further promote taxadiene synthesis, the carbon flux of the GGPP pathway is enhanced by weakening the metabolic flux of the squalene pathway. This is achieved by modulating the promoter P, which regulates squalene synthase transcription. SQS1 Replace with the endogenous weak promoter P of *Yamylostella lipolytica* ERG1 .

[0072] The specific steps are as follows:

[0073] The weak promoter P was amplified from the genome of *Yamanthus lipolyticus* Po1f using primers F23 / R23. ERG1 The progenitor P was amplified from the Po1f genome of *Yamylostella lipolytica* using primers F24 / R24 and F25 / R25. SQS1The 1092bp upper homologous arm and 1025bp lower homologous arm sequences were obtained. The fragment was assembled using OE-PCR. The PCR product was recovered by ethanol precipitation. Approximately 5 μg of the integrated fragment and approximately 1 μg of sgRNA were transformed into the *Yeast Immunoassaycon* engineered strain Tax4 using the Frozen-EZ Yeast Transformation II kit. The transformed strain was plated on screening solid medium and incubated at 30°C for 3 days until colonies appeared. The verified clones were selected and named *Yeast Immunoassaycon* Tax6. The obtained engineered strain Tax6 was cultured in YPD medium with 10% dodecane added and fermented at 30°C and 220 rpm for 120 h. The product content was detected by GC-MS. The results showed that the weakening of squalene helped increase the yield of taxadiene, and the original promoter P... SQS1 Replace with P ERG1 Subsequently, the constructed strain Tax6 produced 58.7±3.8 mg / L of taxadiene, which is currently the strain with the highest taxadiene yield in shake-flask fermentation of lipolytic acid.

[0074] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A taxadiene-producing engineered *Yamylostella lipolytica* strain, characterized in that, The gene encoding taxadiene synthase, with the nucleotide sequence shown in SEQ ID NO.1, is expressed. TXS And with the following improvements: (1) Integration of endogenous MVA biosynthesis pathway genes at the Trp site; the endogenous MVA biosynthesis pathway genes include tHMG1 , ERG12 , ERG20 and IDI1 ; (2) Integration expression at the IntE-1 site Diaporthe amygdali Multifunctional diterpene synthase gene from which this is derived tPaGGPPs Geraniol geraniol pyrophosphate synthase gene derived from Rhodotorula rubiginosa XdGGPPs ; (3) Using promoters P ERG1 Regulation SQS1 Gene expression.

2. The engineered *Yamylostella lipolytica* strain according to claim 1, characterized in that, Using promoters P TEFin Start TXS Gene expression.

3. The engineered *Yamylostella lipolytica* strain according to claim 2, characterized in that, Use promoter P TEF Start tHMG1 Gene, ERG12 Gene, ERG20 Genes and IDI1 Gene expression.

4. The engineered *Yamylostella lipolytica* strain according to any one of claims 1 to 3, characterized in that, Use promoter P TEF Start XdGGPPs Gene expression, and using promoters P TDH Start tPaGGPPs Gene expression.

5. The engineered *Yamylostella lipolytica* strain according to any one of claims 1 to 4, characterized in that, To knock out Ku70 The starting strain was *Agropyron cristatum*. 。 6. A method for preparing taxadiene, characterized in that, The engineered strain of *Yamylostella lipolytica* according to any one of claims 1 to 5 is cultured in a culture medium, and taxadiene in the fermentation broth is collected.

7. The method according to claim 6, characterized in that, The engineered *Yamylostella lipolytica* strain was fermented in a culture medium at 28–30 °C for at least 72 h.

8. The use of the engineered *Yamylostella lipolytica* strain according to any one of claims 1 to 5 in the production of taxadiene-containing products in the food, pharmaceutical, or chemical industries.

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