Nucleotide fragment combination for constructing paclitaxel biosynthesis engineering strain and engineering strain construction method

Abstract

The invention discloses a nucleotide fragment combination for constructing a paclitaxel biosynthesis engineering strain and an engineering strain construction method, and relates to the technical field of bioengineering, the nucleotide fragment combination comprises a nucleotide fragment I, a nucleotide fragment II and a nucleotide fragment III; each fragment respectively comprises genes required by the biological synthesis pathway of 5a-hydroxylated taxadiene, baccatin III and paclitaxel; in each nucleotide fragment, a single promoter is at most used for starting four genes, and the genes started by the single promoter are connected through 2A peptides. The invention also discloses a method for constructing the paclitaxel biosynthesis engineering strain and the paclitaxel biosynthesis engineering strain. According to the invention, the nucleotide fragment combination is utilized to successfully construct the taxol biosynthesis engineering strain of the taxiphocephalum leptosphaeroides through iterative transformation, so that stable biosynthesis of the taxol in a heterologous chassis is realized, and the method has important practical application value.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a combination of nucleotide fragments for constructing paclitaxel engineered strains and a method for constructing engineered strains. Background Technology

[0002] Paclitaxel is a tetracyclic diterpenoid natural compound widely used in the clinical treatment of various cancers. Initially, it could only be extracted from the rare and endangered gymnosperm, the yew. Currently, it is mainly prepared through a semi-synthetic method, based on the taxane intermediates 10-deacetylbaccatin III (10-DAB) and baccatin III extracted from yew branches and leaves, which are then chemically converted to produce paclitaxel. However, the semi-synthetic method still requires scarce yew resources, resulting in high costs and low yields, making it difficult to meet the increasingly severe international demands for cancer prevention and control, and it is also highly likely to cause ecological damage and farmland occupation.

[0003] With the development of synthetic biology and genome sequencing, the genomes of many yew trees have been analyzed, making the biosynthesis of paclitaxel a promising solution. Therefore, constructing engineered biosynthetic strains of paclitaxel holds promise for addressing the growing demand for paclitaxel pharmaceutical raw materials, and has significant scientific and practical application value. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a nucleotide fragment combination for constructing a paclitaxel biosynthetic engineered strain and a method for constructing the engineered strain. This invention utilizes this nucleotide fragment combination to successfully construct a paclitaxel engineered strain through iterative transformation, thereby realizing the biosynthesis of paclitaxel and possessing significant practical application value.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a combination of nucleotide fragments for constructing paclitaxel engineered strains is provided, including nucleotide fragment I, nucleotide fragment II and nucleotide fragment III; Nucleotide fragment I includes GGPPS with the signal peptide removed, TXS with the signal peptide removed, T5αOH, Epoxidase, and the HMGR gene; Nucleotide fragment II includes the genes for T10βOH, T13αOH, DBAT, TAT, TBT, T9αOH, T9α oxidase, T7βOH, CPR, T1βOH, and T2αOH; Nucleotide fragment III includes the BAPT, PCL, PAM, DBTNBT, and TB506 genes; Each nucleotide fragment can be used to start up to 4 genes by a single promoter, and the genes started by a single promoter are linked by a 2A peptide.

[0006] Furthermore, antibiotic resistance selection marker genes are attached to the ends of nucleotide fragments I, II, and III. Furthermore, the nucleotide sequences of nucleotide fragments I, II, and III are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively (the nucleotide sequences have been modified using chassis species limited codons).

[0007] Furthermore, nucleotide fragment I is composed of pACTIN, GGPPS (without signal peptide), P2A, TXS (without signal peptide), P2A, T5αOH, NOS terminator, pAtUBI, Epoxidase, P2A, HMGR, TrbcS, and NPTII linked sequentially. Nucleotide fragment II is composed of pZmUBQ, T10βOH, P2A, T13αOH, P2A, DBAT, P2A, TAT, NOS terminator, pEF1-a, TBT, P2A, T9αOH, P2A, T9α oxidase, TrbcS, p35S promoter, T7βOH, P2A, CPR, TrbcS, pSynA3, T1βOH, P2A, T2αOH, NOS terminator and Hyg linked together in sequence; Nucleotide fragment III is composed of PTA 5' homologous arm, pSynZ9, BAPT, P2A, PCL, P2A, PAM, NOSterminator, pSynZ1, DBTNBT, P2A, TB506, TrbcS terminator and Zeo linked sequentially.

[0008] The present invention also provides a method for constructing a paclitaxel engineered strain, which involves transforming plant protoplasts using the nucleotide fragment combination described above for constructing the paclitaxel engineered strain.

[0009] Furthermore, the aforementioned plants are *Sphaerocarpus septemlobus* or other basal organisms.

[0010] Furthermore, nucleotide fragment I, nucleotide fragment II, and nucleotide fragment III were sequentially transformed through iterative construction.

[0011] The present invention also provides a paclitaxel engineered strain constructed by the above-described method for constructing a paclitaxel biosynthetic engineered strain.

[0012] This invention also provides the application of the above-mentioned paclitaxel engineered strain in the preparation of paclitaxel.

[0013] The present invention has the following beneficial effects: 1. Some genes required for the paclitaxel biosynthesis pathway are toxic to Escherichia coli and yeast, making them difficult to construct. The nucleotide fragment combination of this invention facilitates construction using a low-copy vector.

[0014] 2. Direct transformation of GGPPS, TXS, and T5αOH into *Phyllostachys pubescens* protoplast culture failed to directly produce 5α-hydroxytaxadiene, a key intermediate in the paclitaxel biosynthesis pathway, in *Phyllostachys pubescens*. This is because GGPPS and TXS are located in chloroplasts, while T5αOH is located in the endoplasmic reticulum. The amount of taxadiene produced transported into the cytoplasm is limited, making it difficult to participate in the T5αOH-catalyzed hydroxylation reaction. Therefore, this invention removes the signal peptide from GGPPS and TXS and co-expresses them with T5αOH, successfully detecting 5α-hydroxytaxadiene in the transformed strains.

[0015] 3. The paclitaxel biosynthesis pathway requires a large number of genes, but the promoters currently available for *Pseudomonas aeruginosa* are limited. This invention reduces the use of promoters by tandem multiple genes with a 2A peptide.

[0016] 4. Constructing the complete paclitaxel biosynthetic pathway onto a single vector results in large DNA fragments, and errors are difficult to correct. This invention employs a multi-iterative construction method, dividing the entire paclitaxel biosynthetic pathway into three parts: 5α-hydroxytaxadiene, baccatin III, and paclitaxel. These parts are sequentially transformed to construct engineered strains, allowing for the distribution and verification of paclitaxel and its intermediates. Furthermore, because paclitaxel and its intermediates can negatively impact the growth of *Phyllostachys edulis* cells, the transformation efficiency for constructing paclitaxel engineered strains is extremely low. This invention's method facilitates verification of transformation effects and improves transformation efficiency. Attached Figure Description

[0017] Figure 1 The image shows the detection results of 5α-hydroxytaxadiene; Figure 2 This is a spectral analysis result of paclitaxel standard. Figure 3 This is a graph showing the mass spectrometry analysis results of the culture products of the transformed strain. Detailed Implementation

[0018] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0019] The following are some of the materials used in the implementation process. MMM solution: 9.1% Mannitol, 0.015 mmol / L MgCl2, 0.1% MES.

[0020] PRMB solution: 1.84 mmol / L KH2PO4, 1 mmol / L MgSO4·7H2O, 40 mmol / L KOH, 10 mM KNO3, 45 µmol / L FeSO4·7H2O, 5.0 mmol / L ammonium tartrate, 0.22 µmol / L CuSO4·5H2O, 10 µmol / L H3BO3, 0.23 µmol / L CoCl2·6H2O, 0.19 µmol / L ZnSO4·7H2O, 2 µmol / L MnCl2·4H2O, 0.17 µmol / L KI, 0.1 µmol / L Na2MoO4·2H2O, 10 mmol / L CaCl2·2H2O, 0.48 M mannitol, 0.3% Glucose, and 0.8% Agar.

[0021] PRMT plate: 1.84 mmol / L KH2PO4, 1 mmol / L MgSO4·7H2O, 40 mmol / L KOH, 10 mM KNO3, 45 µmol / L FeSO4·7H2O, 5.0 mmol / L ammonium tartrate, 0.22 µmol / L CuSO4·5H2O, 10 µmol / L H3BO3, 0.23 µmol / L CoCl2·6H2O, 0.19 µmol / L ZnSO4·7H2O, 2 µmol / L MnCl2·4H2O, 0.17 µmol / L KI, 0.1 µmol / L Na2MoO4·2H2O and 0.48 M mannitol, 10 mmol / L CaCl2·2H2O 0.3% Glucose, 10 mmol / L 0.6% Agar.

[0022] BCD medium: 1 mmol / L MgSO4·7H2O, 1.84 mmol / L KH2PO4, 10 mmol / L KNO3, 0.045 mmol / L FeSO4·7H2O, 0.22 μmol / L CaSO4·5H2O, 10 μmol / L H3BO3, 0.23 μmol / L CoCL2·6H2O, 0.1 μmol / L NaMoO4·2H2O, 0.19 μmol / L ZnSO4·7H2O, 2 μmol / L MnCl2·4H2O, 0.17 μmol / L KI, 1 mmol / L CaCl2·2H2O, 0.75% Agar.

[0023] BCDAT medium: 1 mmol / L MgSO4·7H2O, 1.84 mmol / L KH2PO4, 10 mmol / L KNO3, 0.045 mmol / L FeSO4·7H2O, 0.22 μmol / L CaSO4·5H2O, 10 μmol / L H3BO3, 0.23 μmol / LCoCL2·6H2O, 0.1 μmol / L NaMoO4·2H2O, 0.19 μmol / L ZnSO4·7H2O, 2 μmol / L MnCl2·4H2O, 0.17 μmol / L KI, 5 mmol / L Ammonium Tartrate, 1 mmol / L CaCl2·2H2O, 0.75% Agar.

[0024] Example 1 A combination of nucleotide fragments for constructing a paclitaxel biosynthetic engineered strain, comprising nucleotide fragment I, nucleotide fragment II, and nucleotide fragment III; The nucleotide fragment I, as shown in SEQ ID NO.1, is composed of pACTIN, GGPPS (with signal peptide removed), P2A, TXS (with signal peptide removed), P2A, T5αOH, NOS terminator, pAtUBI, Epoxidase, P2A, HMGR, TrbcS, and NPTII linked together in sequence. Nucleotide fragment II, as shown in SEQ ID NO.2, is composed of pZmUBQ, T10βOH, P2A, T13αOH, P2A, DBAT, P2A, TAT, NOS terminator, pEF1-a, TBT, P2A, T9αOH, P2A, T9α oxidase, TrbcS, p35S promoter, T7βOH, P2A, CPR, TrbcS, pSynA3, T1βOH, P2A, T2αOH, NOS terminator, and Hyg linked sequentially. Nucleotide fragment III, as shown in SEQ ID NO.3, is composed of PTA 5' homologous arm, pSynZ9, BAPT, P2A, PCL, P2A, PAM, NOS terminator, pSynZ1, DBTNBT, TB506, TrbcS terminator, and Zeo linked sequentially.

[0025] Example 2 Genetic transformation and screening of engineered strains of *Bryum simulans* (1) Nucleotide fragment I, nucleotide fragment II and nucleotide fragment III in Example 1 were designed as multiple fragments of less than 3 kb and chemically synthesized, and then assembled by Saccharomyces cerevisiae to obtain nucleotide fragment I, nucleotide fragment II and nucleotide fragment III; (2) Take the material from the protonema stage of *Scutellaria baicalensis* about five days after the second subculture, lyse it with 20 mL of lysin solution, let it stand at room temperature for 30 min, gently mix it at intervals, then filter it through a cell sieve, centrifuge the filtrate at 180 g for 2 min, wash it with 8% D-mannitol solution and resuspend it in an appropriate amount of MMM solution to obtain a concentration of 1.6 × 10⁻⁶. 5 Protoplast weight suspension per mL; (3) Take out about 300 μL of protoplast resuspension, add 10-30 μg of nucleotide fragment I, mix gently and let stand at room temperature for 15 min, add an equal volume of PEGT solution, shake gently and mix well and let stand for 30 min, wash the protoplasts with W5 solution, centrifuge at 180 g and add 9 mL of PRMB to resuspend the protoplasts, pour them onto 3 PRMT plates, and culture at 25℃ for 8 h in the dark / 16 h in the light. After 2 weeks of culture, transfer to a medium containing the corresponding antibiotics for screening to obtain the transformant strain that successfully transforms nucleotide fragment I; (4) Based on steps (1)-(3), transform the transformant strain with nucleotide fragment II to obtain a transformant strain that is simultaneously transformed with nucleotide fragment I and nucleotide fragment II; (5) Based on the transformation strain, transform nucleotide fragment III according to steps (1)-(3) to obtain a transformation strain that successfully transforms the above nucleotide fragment combination.

[0026] Example 3: Transformation Culture and Product Detection The transformants obtained in Example 2 were grown into stem-leaf stalks on BCD medium plates. The stem-leaf stalk material was collected, homogenized, and spread evenly on BCDAT medium for further cultivation. After collecting a sufficient amount of transformant material, the material was dried, methanol was added, and the mixture was placed in an ice-water mixture. Paclitaxel was extracted using an ultrasonic homogenizer (8 s working, 4 s intermittent, total duration 20 min, power 120 W). After centrifugation at 5000 rpm / min for 5 min, the organic phase was collected, filtered through an organic filter membrane, and transferred to a mass spectrometry bottle for detection using a XevoG2-XS Qtof instrument. Simultaneously, transformants without the removal of GGPPS and TXS signal peptides were obtained according to Example 2 and cultured for further analysis.

[0027] (1) Results of 5α-hydroxytaxadiene detection in different transformants are as follows: Figure 1 As shown, Sample 1 is a transformant strain for which GGPPS and TXS signal peptides were not removed, and Sample 2 is a transformant strain from Example 2.

[0028] Depend on Figure 1 It can be seen that after removing the signal peptide from GGPPS and TXS, co-expressing them with T5αOH successfully detected 5α-hydroxytaxane in the transformant.

[0029] (2) Figure 2 Mass spectrum of paclitaxel standard. Figure 3 This is a graph showing the detection results of the culture products from the transformant strain of Example 2. The results show that paclitaxel was detected in the extract of the transformant strain of Example 2.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A combination of nucleotide fragments for constructing a paclitaxel biosynthetic engineered strain, characterized in that, Includes nucleotide fragment I, nucleotide fragment II, and nucleotide fragment III; The nucleotide fragment I includes GGPPS with the signal peptide removed, TXS with the signal peptide removed, T5αOH, Epoxidase, and the HMGR gene; Nucleotide fragment II includes the genes for T10βOH, T13αOH, DBAT, TAT, TBT, T9αOH, T9α oxidase, T7βOH, CPR, T1βOH, and T2αOH; Nucleotide fragment III includes the BAPT, PCL, PAM, DBTNBT, and TB506 genes; Each nucleotide fragment can be used to start up to 4 genes by a single promoter, and the genes started by a single promoter are linked by a 2A peptide.

2. The nucleotide fragment combination for constructing a paclitaxel biosynthetic engineered strain as described in claim 1, characterized in that, Nucleotide fragments I, II, and III are each connected to a selection marker gene at their ends.

3. The nucleotide fragment combination for constructing a paclitaxel biosynthetic engineered strain as described in claim 1, characterized in that, The nucleotide sequences of nucleotide fragment I, nucleotide fragment II, and nucleotide fragment III are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

4. A method for constructing a paclitaxel biosynthetic engineered strain, characterized in that, Transform plant protoplasts using the nucleotide fragment combination for constructing paclitaxel engineered plants as described in claim 1.

5. The method for constructing a paclitaxel biosynthetic engineered strain as described in claim 4, characterized in that, The plant in question is *Moss sphaerocephala*.

6. The method for constructing a paclitaxel biosynthetic engineered strain as described in claim 4, characterized in that, Nucleotide fragment I, nucleotide fragment II, and nucleotide fragment III were sequentially transformed through iterative construction.

7. The paclitaxel biosynthetic engineered strain constructed by the method for constructing paclitaxel biosynthetic engineered strains according to any one of claims 4-6.

8. The use of the paclitaxel biosynthetic engineered strain according to claim 7 in the preparation of paclitaxel.

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