A high-efficiency expression method of a yeast for an intermediate synthesis enzyme of anti-aids

Abstract

This invention discloses a method for efficient yeast expression of an anti-AIDS intermediate synthase, belonging to the field of green synthesis of biopharmaceutical intermediates and enzyme engineering technology. This invention obtains the target gene encoding the ketone reductase / aldehyde-ketone reductase required for the synthesis of darunavir intermediates or HIV protease inhibitor intermediates, optimizes the codons according to the Pichia pastoris preference, and introduces the Kozak-like sequence AAAAAAAATG at positions -12 to -1 upstream of the 5′UTR to the start codon. The optimized gene and the catalytic domain of glucose-6-phosphate dehydrogenase ZWF1 are co-cloned into a Pichia pastoris secretory expression vector, electroporated into GS115 or KM71 strains, and positive transformants are obtained by Zeocin resistance screening and PCR identification. Expression is induced by a methanol gradient in BMMY medium and the fermentation supernatant is collected, centrifuged, and filtered to obtain a highly active soluble crude enzyme solution. This invention significantly enhances the expression level, catalytic activity, stability, and solubility of the synthase through the synergistic effects of codon optimization, translation initiation enhancement, in situ coenzyme regeneration, and mild gradient induction. It can efficiently catalyze the selective reduction of the (S)-configuration of the darunavir intermediate, providing efficient technical support for the green enzymatic preparation of key intermediates in anti-AIDS drugs.

Description

Technical Field

[0001] This invention belongs to the field of green synthesis of biopharmaceutical intermediates and enzyme engineering application technology, specifically relating to a yeast method for efficient expression of an anti-AIDS intermediate synthase. Background Technology

[0002] AIDS (Acute HIV / AIDS) is a serious infectious disease caused by infection with the human immunodeficiency virus (HIV), posing a significant threat to human health. Darunavir and other HIV protease inhibitors, characterized by their potency, broad spectrum, and high resistance barrier, are currently core drugs in clinical treatment of AIDS. Ketoreductase and aldosterone reductase are key catalytic enzymes in the synthesis of darunavir intermediates and other HIV protease inhibitor intermediates. Their activity and expression levels directly determine the synthesis efficiency, conformational selectivity, and product purity of the intermediates. Efficient and highly selective synthesis of intermediates is crucial for reducing drug production costs and improving drug accessibility.

[0003] Currently, the preparation of anti-AIDS intermediate synthases mainly relies on prokaryotic and yeast expression systems. Prokaryotic expression systems suffer from drawbacks such as the easy formation of inclusion bodies in the expressed products, poor solubility, insufficient activity, and lack of post-translational modifications. These characteristics lead to low catalytic efficiency and poor stability of the synthases, making it difficult to meet the demands of industrial production. Pichia pastoris, as a eukaryotic expression host, offers advantages such as high expression levels, correct post-translational modifications, secretion of target proteins, low culture costs, and strong environmental adaptability, and has been widely used in the efficient preparation of industrial enzymes and pharmaceutical proteins.

[0004] While the Pichia pastoris system offers significant advantages, the natural ketone reductase and aldehyde-ketone reductase genes in Pichia pastoris still suffer from problems such as excessively high rare codon frequencies, low translation initiation efficiency, and limited secretion efficiency. This results in low expression levels and inconsistent activity of the synthases, and some expression systems lack NADPH regeneration support, further limiting the catalytic efficiency of the synthases. Furthermore, unreasonable induction conditions and poor product stability in existing expression methods also hinder the large-scale application of anti-AIDS intermediate synthesis. Therefore, developing an efficient Pichia pastoris expression method for anti-AIDS intermediate synthases, through codon optimization, expression vector improvement, and induction strategy optimization to achieve high-activity, high-soluble, and high-stability expression of the synthases, has significant theoretical and industrial application value.

[0005] Patent CN118165948A discloses a method for high expression of ketone reductase in Pichia pastoris and its application. It uses an in vitro construction method to construct a multi-copy expression recombinant vector to obtain high copy number recombinant bacteria, which eliminates the need for extensive screening to obtain high copy strains, greatly reducing the workload. The recombinant ketone reductase has high enzyme activity. However, this invention does not perform any codon optimization on the coding gene, and directly uses the fixed nucleotide sequence SEQ ID NO.1 for expression, without considering the codon bias of Pichia pastoris, resulting in low gene translation efficiency and easy translation stagnation.

[0006] In summary, there is an urgent need for a method to optimize the coding genes in Pichia pastoris using any codons to improve the expression level, catalytic activity, and storage stability of the synthase. Summary of the Invention

[0007] The purpose of this invention is to provide a method for efficient yeast expression of an anti-AIDS intermediate synthase, in order to solve the problems mentioned in the background art.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution: a method for efficient yeast expression of an anti-AIDS intermediate synthase, comprising the following steps: S1: Obtain the target gene encoding the ketone reductase or aldehyde-ketone reductase required for the synthesis of darunavir intermediate or HIV protease inhibitor intermediate; S2: Codon optimization is performed on the target gene obtained in S1, and a Kozak-like sequence AAAAATG is introduced by 5′UTR to position -12 to -1 upstream of the start codon; S3: The optimized gene obtained in S2 was cloned into the Pichia pastoris secretory expression vector; S4: The recombinant vector obtained in S3 was electroporated into Pichia pastoris competent cells, plated on YPD solid medium containing Zeocin (100 μg / mL), and cultured at 30 ℃ for 48 h. Single colonies were picked for PCR identification to obtain positive transformants. S5: Inoculate positive transformants into BMGY medium and incubate at 28-30 ℃ with shaking at 250 r / min until OD. 600 =2-6, transferred to BMMY induction medium, and induced by methanol gradient: first maintain 0.5% methanol for 24 h, then increase to 1.0% methanol volume percentage and maintain for 24 h, and finally increase to 1.5% methanol volume percentage and maintain for 24-48 h. S6: Collect the fermentation supernatant, centrifuge at 8000×g for 15 min to remove the bacterial cells, and then filter the supernatant through a 0.22 μm filter membrane to obtain a crude enzyme solution containing highly active soluble synthetic enzymes.

[0009] Using *Saccharomyces cerevisiae* as the expression host, codon optimization was performed on the anti-AIDS intermediate synthase gene. The 5Kozal-like sequence AAAAAAATG was introduced into the 5′UTR region to enhance ribosome binding and recognition capabilities, improve translation initiation and elongation efficiency, and increase protein expression and solubility from the source.

[0010] Furthermore, in S2, the strain of Pichia pastoris is either GS115 or KM71.

[0011] Furthermore, in S3, the Pichia pastoris secretory expression vector is pPICZαA or pPIC9k.

[0012] Furthermore, in S3, the Pichia pastoris secretory expression vector includes: an AOX1 promoter, an α-factor signal peptide coding sequence, a multiple cloning site, and a Zeocin resistance gene.

[0013] Furthermore, in S3, the α-factor signal peptide is linked to the 5′ end of the optimized gene, with the cleavage site being Lys-Arg.

[0014] The optimized gene was cloned into the pPICZαA / pPIC9k secretory expression vector. The AOX1 promoter drove the transcription of the exogenous gene, and the α-factor signal peptide encoding sequence carried by the vector guided the extracellular secretion of the target protein. After being cleaved at the Lys-Arg cleavage site, the mature protein was released, simplifying the downstream separation and purification process.

[0015] Furthermore, in S3, the expression vector also contains a catalytic domain encoding fragment of glucose-6-phosphate dehydrogenase ZWF1, with the amino acid sequence being positions 1-420 of SEQ ID NO. 5.

[0016] Furthermore, the particle size D of the synthesized enzyme in the crude enzyme solution 50 =12-18 nm, and the Zeta potential is +28- +32 mV at pH 7.2 and 5 ℃.

[0017] Furthermore, the crude enzyme solution is used for the selective reduction of the (S)-configuration of the darunavir intermediate, and the reaction system includes a substrate, glucose, and crude enzyme solution.

[0018] Furthermore, the substrate concentration in the reaction system is 10-50 g / L, the glucose concentration is 20-60 g / L, and the protein concentration in the crude enzyme solution is 0.5-2.0 g / L.

[0019] Furthermore, the crude enzyme solution is freeze-dried to obtain lyophilized powder, and the enzyme activity recovery rate after reconstitution is ≥92%.

[0020] The beneficial effects of this invention are: 1. This invention performs precise codon optimization on the target gene based on the codon preference of Pichia pastoris, controls the frequency of rare codons in Pichia pastoris to ≤0.5%, introduces the Kozak-like sequence AAAAAAATG into the translation initiation region, improves cassette recognition and binding efficiency, and significantly improves gene translation efficiency and synthase expression level; and selects Pichia pastoris strains GS115 or KM71 to avoid the bottleneck of single strains.

[0021] 2. This invention uses pPICZαA or pPIC9k secretory expression vectors, and the α-factor signal peptide can guide the efficient extracellular secretion of the synthase; the introduction of the ZWF1 catalytic domain encoding fragment can regenerate NADPH in situ, achieving coenzyme self-sufficiency and further improving the activity and stability of the synthase.

[0022] 3. This invention uses electroconversion to improve the transformation efficiency of recombinant vectors, and combines Zeocin resistance screening and PCR identification to ensure the purity of positive transformants. It adopts a two-step fermentation strategy of BMGY amplification culture and BMMY gradient methanol induction to avoid methanol toxicity and precisely control the expression level. The promoter is continuously activated at 28-30℃ to achieve highly soluble secretory expression of the target protein.

[0023] Table 1 is a summary table of the test results of the embodiments and comparative examples of the present invention. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the appendix.

[0025] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

[0026] Example 1: S1: The KpADH gene was artificially synthesized. The gene is 1074 bp in length, and its nucleotide sequence corresponds to GenBank accession number MK123456.1. At the same time, the catalytic domain fragment of glucose-6-phosphate dehydrogenase ZWF1 (hereinafter referred to as ZWF1cd) was artificially synthesized. The fragment is 1260 bp in length, and its encoded amino acid sequence is positions 1-420 of SEQ ID NO.5. BamHI and XhoI restriction endonuclease sites were added to both ends of the above KpADH gene and ZWF1cd fragment. After the synthesized gene fragments were verified to be correct by sequencing, they were stored at -20℃ for later use.

[0027] S2: Using the KpADH gene synthesized in S1 as a template, the KpADH gene full sequence was codon optimized according to the codon preference of Pichia pastoris strain GS115. A Kozak-like sequence AAAAAAAATG was inserted into the 5′ UTR end of the optimized KpADH gene. After the optimized gene was amplified by PCR, purified and sequenced to verify that it was correct, the optimized KpADH gene was obtained.

[0028] S3: The optimized KpADH gene obtained in S2 and the ZWF1cd fragment synthesized in S1 were double-digested using BamHI and XhoI restriction endonucleases (Takara, 10 U / μL). Specific digestion parameters were as follows: 50 μL digestion system, containing 20 μL of the target gene (optimized KpADH gene or ZWF1cd fragment), 2 μL of BamHI, 2 μL of XhoI, 5 μL of 10× digestion buffer (Takara, corresponding to the universal buffer for BamHI and XhoI), and 21 μL of enzyme-free ddH2O; the digestion conditions were incubation at 37℃ for 3 days. h. After enzyme digestion, the digestion products were purified and recovered using a DNA purification kit (Omega) to remove the digestion buffer and other enzymes. The purified optimized KpADH gene and ZWF1cd fragment were co-cloned into the multiple cloning site (MCS) of the Pichia pastoris secretory expression vector pPICZαA to construct a recombinant expression vector named pPICZαA-KpADH-ZWF1cd. After construction, the recombinant vector was identified by enzyme digestion and verified by Sanger sequencing to confirm that the optimized KpADH gene and ZWF1cd fragment were correctly inserted into the vector.

[0029] S4: The recombinant expression vector pPICZαA-KpADH-ZWF1cd constructed in S3 was introduced into Pichia pastoris GS115 competent cells by electroporation. After electroporation, the bacterial culture was plated on YPD solid medium containing Zeocin (concentration of 100 μg / mL) and incubated in a constant temperature incubator at 30 ℃ for 48 h. After single colonies appeared on the plate, single colonies were picked for PCR verification. The primers used for PCR verification were: upstream primer F: 5′-TTACGTTCAGTTTAGCTGG-3′, downstream primer R: 5′-GCTGCCAGCCAAACTTGT-3′. The PCR-positive colonies were further sequenced to confirm that the target gene insertion was correct and the sequence was error-free, and positive transformants were obtained.

[0030] S5: Pick the positive transformants obtained in S4, inoculate them into BMGY medium, and incubate with shaking at 30 ℃ and 250 r / min until the bacterial culture reaches OD. 600When the value reached 4.2, the seed culture was obtained. The seed culture was then transferred to BMMY induction medium and induced expression was carried out using a methanol gradient induction strategy: first, the culture was maintained at a methanol volume percentage concentration of 0.5% (v / v) for 24 h, then the methanol volume percentage concentration was increased to 1.0% (v / v) for 24 h, and finally the methanol volume percentage concentration was increased to 1.5% (v / v) for 24 h. The temperature was controlled at 28 ℃ throughout the fermentation induction process to ensure sufficient dissolved oxygen.

[0031] S6: After fermentation induction, the supernatant of the fermentation broth was collected and placed in a high-speed centrifuge. The supernatant was centrifuged at 8000×g for 15 min to remove the cell precipitate. The supernatant after centrifugation was filtered through a 0.22 μm aqueous filter membrane to remove impurities, and finally a clear brownish-yellow crude enzyme solution was obtained. The protein concentration of the crude enzyme solution was found to be 1.2 g / L.

[0032] Example 2 S1: The target gene encoding aldehyde-ketone reductase was artificially synthesized, with EcoRI and NotI restriction sites added at both ends, and sequencing confirmed its correctness.

[0033] S2: The codon preference of Pichia pastoris strain KM71 was optimized to make the frequency of rare codons ≤0.5%, and the Kozak-like sequence AAAAAAAATG was introduced from the 5′UTR to the position -12 to -1 upstream of the start codon.

[0034] S3: The optimized gene was cloned into the secretory vector pPIC9k, which contains the AOX1 promoter, α-factor signal peptide, and His4 tag.

[0035] S4: The recombinant vector was electroporated into KM71 competent cells, plated on YPD plates containing G418, and cultured at 30 ℃ for 48 h. Positive transformants were obtained by PCR identification.

[0036] S5: Positive transformants were inoculated into BMGY cells and cultured at 30 ℃ and 250 r / min until OD600=3.8. They were then transferred to BMMY cells for methanol gradient induction. The methanol volume percentage concentration gradient was: 0.5% (v / v) 24 h → 1.0% (v / v) 24 h → 1.5% (v / v) 36 h, with the entire process at 28 ℃. 6. Centrifuge the fermentation broth at 8000×g for 15 min, filter through a 0.22 μm filter membrane to obtain a clear crude enzyme solution. Comparative Example 1 S1: Artificial Synthesis of Gene Fragments: The natural KpADH gene, which has not undergone codon optimization, was artificially synthesized. The gene is 1074 bp in length, and its nucleotide sequence corresponds to GenBank accession number MK123456.1. At the same time, the catalytic domain fragment (ZWF1cd) of glucose-6-phosphate dehydrogenase ZWF1 was artificially synthesized. The fragment is 1260 bp in length, and its encoded amino acid sequence is positions 1-420 of SEQ ID NO.5. BamHI and XhoI restriction endonuclease sites were added to both ends of the above-mentioned natural KpADH gene and ZWF1cd fragment. After the synthesized gene fragments were verified to be correct by sequencing, they were stored at -20 ℃ for later use.

[0037] S2: Using the natural KpADH gene synthesized in S1 as a template, without codon optimization, and without inserting the Kozak-like sequence AAAAAATG into the 5′UTR region; the natural KpADH gene is used after being amplified by PCR, purified and sequenced to verify that it is correct.

[0038] S3: The natural KpADH gene obtained in S2 and the ZWF1cd fragment synthesized in S1 were double-digested using BamHI and XhoHI restriction endonucleases, respectively. The digestion system was 50 μL, containing 20 μL of the target gene, 2 μL of BamHI, 2 μL of XhoHI, 5 μL of 10× digestion buffer, and 21 μL of enzyme-free ddH2O. The digestion reaction was carried out at 37 ℃ for 3 h. After digestion, the digestion products were purified and recovered. The purified natural KpADH gene and ZWF1cd fragment were co-cloned into the multiple cloning site of the Pichia pastoris secretory expression vector pPICZαA to construct the recombinant expression vector pPICZαA-KpADH-ZWF1cd. The digestion and sequencing confirmed that the results were correct.

[0039] S4: The recombinant expression vector pPICZαA-KpADH-ZWF1cd was electroporated into Pichia pastoris GS115 competent cells; it was plated on YPD solid medium containing Zeocin (100 μg / mL) and cultured at 30 ℃ for 48 h. Single colonies were picked for PCR and sequencing verification to obtain positive transformants.

[0040] S5: Induction of expression: Positive transformants were picked and inoculated into BMGY medium, and cultured at 30 ℃ with shaking at 250 r / min until OD500 was reached. 600 =4.2, transferred to BMMY induction medium, and induced by methanol gradient: 0.5% (v / v) methanol for 24 h, 1.0% (v / v) methanol for 24 h, 1.5% (v / v) methanol for 24 h, and the temperature was controlled at 28 ℃ throughout.

[0041] S6: Preparation of crude enzyme solution: Collect the fermentation supernatant, centrifuge at 8000×g for 15 min to remove the cells, and filter the supernatant through a 0.22 μm filter membrane to obtain crude enzyme solution.

[0042] Comparative Example 2 The uncodon-optimized natural ketone reductase gene was cloned into the ordinary pPICZαA vector, which does not contain the ZWF1 catalytic domain. It was then transformed into GS115 and continuously induced with a constant 1.0% methanol for 72 h without gradient regulation. The steps for preparing the crude enzyme solution were the same as in Example 1. Table 1 As shown in Table 1, Examples 1 and 2, employing the codon optimization, Kozak-like sequence introduction, ZWF1 catalytic domain co-expression, and methanol gradient induction strategies described in this invention, achieved high levels of crude enzyme protein concentration, relative enzyme activity, enzymatic stability, and protein solubility, with comparable results between the two groups. Comparative Example 1, which lacked codon optimization, had a crude enzyme protein concentration of only 0.52 g / L, a relative enzyme activity only 43% of Example 1, and significantly reduced stability and solubility. Comparative Example 2, using a conventional method without optimized natural genes, Kozak-like sequences, or ZWF1 catalytic domain and constant methanol induction, showed significantly worse performance than the examples, with a crude enzyme protein concentration of only 0.31 g / L and a relative enzyme activity only 24% of Example 1, exhibiting severe protein aggregation and poor stability.

[0043] The above results demonstrate that the present invention, through the synergistic effect of codon optimization, Kozak-like sequence, co-expression of the ZWF1 catalytic domain, and methanol gradient induction, can significantly improve the expression level, catalytic activity, stability, and solubility of anti-AIDS intermediate synthase in Pichia pastoris, with significantly better results than traditional expression methods.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for efficient yeast expression of an anti-AIDS intermediate synthase, characterized in that, Includes the following steps: S1: Obtain the target gene encoding the ketone reductase or aldehyde-ketone reductase required for the synthesis of darunavir intermediate or HIV protease inhibitor intermediate; S2: Codon optimization is performed on the target gene obtained in S1, and a Kozak-like sequence AAAAATG is introduced by 5′UTR to position -12 to -1 upstream of the start codon; S3: The optimized gene obtained in S2 was cloned into the Pichia pastoris secretory expression vector; S4: The recombinant vector obtained in S3 was electroporated into Pichia pastoris competent cells, plated on YPD solid medium containing Zeocin (100 μg / mL), and cultured at 30 ℃ for 48 h. Single colonies were picked for PCR identification to obtain positive transformants. S5: Inoculate positive transformants into BMGY medium and incubate at 28-30 ℃ with shaking at 250 r / min until OD. 600 =2-6, transferred to BMMY induction medium, and induced by methanol gradient: first maintain 0.5% methanol for 24 h, then increase to 1.0% methanol volume percentage and maintain for 24 h, and finally increase to 1.5% methanol volume percentage and maintain for 24-48 h. S6: Collect the fermentation supernatant, centrifuge at 8000×g for 15 min to remove the bacterial cells, and then filter the supernatant through a 0.22 μm filter membrane to obtain a crude enzyme solution containing highly active soluble synthetic enzymes.

2. The method for high-efficiency yeast expression of the anti-AIDS intermediate synthase according to claim 1, characterized in that, In S2, the strain of Pichia pastoris is either GS115 or KM71.

3. The method for high-efficiency yeast expression according to claim 1, characterized in that, In S3, the secretory expression vector of Pichia pastoris is pPICZαA or pPIC9k.

4. The method for high-efficiency yeast expression of the anti-AIDS intermediate synthase according to claim 1, characterized in that, In S3, the Pichia pastoris secretory expression vector contains: an AOX1 promoter, an α-factor signal peptide coding sequence, a multiple cloning site, and a Zeocin resistance gene.

5. The method for high-efficiency yeast expression of the anti-AIDS intermediate synthase according to claim 1, characterized in that, In S3, the α-factor signal peptide is linked to the 5′ end of the optimized gene, and the cleavage site is Lys-Arg.

6. The method for high-efficiency yeast expression of the anti-AIDS intermediate synthase according to claim 1, characterized in that, In S3, the expression vector also contains a catalytic domain encoding fragment of glucose-6-phosphate dehydrogenase ZWF1, with the amino acid sequence being positions 1-420 of SEQ ID NO.

5.

7. The method for high-efficiency yeast expression of the anti-AIDS intermediate synthase according to claim 1, characterized in that, The particle size D of the synthesized enzyme in the crude enzyme solution 50 =12-18 nm, pH7.2, Zeta potential at 5 ℃ is +28- +32 mV.

8. The method for high-efficiency yeast expression of the anti-AIDS intermediate synthase according to claim 1, characterized in that, The crude enzyme solution is used for the selective reduction of the (S)-configuration of darunavir intermediate. The reaction system includes substrate, glucose, and crude enzyme solution.

9. The method for high-efficiency yeast expression of the anti-AIDS intermediate synthase according to claim 8, characterized in that, The substrate concentration in the reaction system is 10-50 g / L, the glucose concentration is 20-60 g / L, and the protein concentration in the crude enzyme solution is 0.5-2.0 g / L.

10. The method for high-efficiency yeast expression of the anti-AIDS intermediate synthase according to any one of claims 8 to 9, characterized in that, The crude enzyme solution was freeze-dried to obtain freeze-dried powder, and the enzyme activity recovery rate after reconstitution was ≥92%.

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