A recombinant pichia pastoris strain, a preparation method and application thereof

Abstract

The application discloses a recombinant Pichia pastoris strain and a preparation method and application thereof, and relates to the technical field of biology. The preparation method of the recombinant Pichia pastoris strain comprises the following steps: constructing a promoter and a 2A peptide element library, introducing an R3GAT gene into a Pichia pastoris genome, excavating and knocking out an endogenous beta-glucosidase gene, and then optimizing and integrating a UDP-glucose supply module gene, so that the recombinant Pichia pastoris strain is obtained. The application also provides application of the recombinant Pichia pastoris strain in production of rhizomaliside, and effectively solves the problems of low yield of rhizomaliside due to degradation of endogenous glycosidase in Pichia pastoris and low synthesis efficiency caused by insufficient supply of UDP-glucose.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant Pichia pastoris strain, its preparation method, and its application. Background Technology

[0002] Natural products, especially plant secondary metabolites, are frequently used in the production of pharmaceuticals, nutritional supplements, and cosmetics due to their diverse biological activities, including antimalarial, anticoagulant, antitumor, antioxidant, anti-aging, and anti-inflammatory properties. The production methods for plant-derived natural compounds mainly include four strategies: natural product extraction, chemical synthesis, enzymatic catalysis, and microbial synthesis. Natural product extraction is the primary method for producing plant-derived active ingredients, but it suffers from drawbacks such as high land resource consumption, low extraction yield, and low concentrations of active ingredients in the plant. Chemical synthesis faces challenges such as high toxicity of organic solvents, complex synthesis processes, and poor food safety. Enzymatic catalysis requires the addition of expensive cofactors to participate in the catalytic reaction. Microbial synthesis is the main method for producing plant-derived active ingredients, characterized by its natural, green, safe, and sustainable nature. Microbial synthesis is also a primary method for producing plant-derived natural glycosides.

[0003] Glycosides are a class of organic molecules that are widely found in nature, consisting of a sugar moiety and a non-sugar moiety (glycoside) linked by glycosidic bonds. Their structural diversity and biological activity make them important secondary metabolites in plants, microorganisms, and even some animals. They include simple monosaccharide glycosides such as arbutin and senna glycoside, as well as complex polysaccharide chain glycosides such as saponins, flavonoid glycosides, and cardiac glycosides. They usually have significant water solubility, stability, and specific pharmacological activities.

[0004] Polygonum cuspidatum glycoside, also known as resveratrol glycoside, can be converted into resveratrol in vivo, thus exerting similar biological effects. Polygonum cuspidatum glycoside is often considered a precursor or analogue of resveratrol. It is also a naturally occurring polyphenolic glycoside compound, possessing multi-target, low-toxicity, and broad-spectrum activity, along with a wide range of pharmacological activities including anti-aging, antioxidant, anti-tumor, anti-inflammatory, cardiovascular protection, and other effects. Due to the poor water solubility and low stability of resveratrol itself, 3-O-glycosylation of it to obtain polygonum cuspidatum glycoside not only improves water solubility but also significantly enhances oral bioavailability and in vivo stability, leading to increasing demand in functional foods, cosmetics, and innovative pharmaceuticals. Summary of the Invention

[0005] To address the aforementioned shortcomings in the existing technology, this invention provides a recombinant Pichia pastoris strain, its preparation method, and its application, effectively solving the problems of low yield of polygalactosidase in Pichia pastoris due to degradation by endogenous glycosidases and low synthesis efficiency due to insufficient UDP-glucose supply.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is: to provide a method for preparing a recombinant Pichia pastoris strain, comprising the following steps: constructing a promoter and 2A peptide element library, introducing the R3GAT gene into the Pichia pastoris genome, mining and knocking out the endogenous β-glucosidase gene, and then optimizing and integrating the UDP-glucose supply module gene, thereby obtaining a recombinant Pichia pastoris strain.

[0007] Furthermore, the Pichia pastoris is Pichia pastoris GS115.

[0008] Furthermore, the R3GAT gene achieves multi-gene co-expression through the 2A peptide.

[0009] Furthermore, peptide 2A is OpbuCPV18 or EMCV.

[0010] Furthermore, the endogenous β-glucosidase gene is the 891B gene or / and the EXG1 gene.

[0011] Furthermore, the UDP-glucose supply module contains at least one of the YNK1, PGM2, and galU genes.

[0012] Furthermore, the genes in the UDP-glucose supply module are expressed by constitutive promoters.

[0013] Furthermore, the constitutive promoter is at least one of pTDH3, pGAP, and pGCW14.

[0014] Furthermore, the UDP-glucose supply module gene is inserted into the pGAP site through tandem integration or iterative integration, and its expression is driven by the pTEF1 promoter.

[0015] The recombinant Pichia pastoris strain was prepared using the above method.

[0016] The above-mentioned recombinant Pichia pastoris strain is used in the production of polygalactoside or in food, health products, cosmetics or pharmaceuticals containing polygalactoside.

[0017] A method for producing polydipsia glycoside includes the following steps: culturing a recombinant Pichia pastoris strain, adding resveratrol to a fermentation medium for fermentation, and collecting polydipsia glycoside.

[0018] Furthermore, the fermentation medium is one of BMTY medium, BMGY medium and BMMY medium.

[0019] Furthermore, the fermentation medium was BMTY medium.

[0020] Furthermore, the concentration of resveratrol added is 2-6 g / L.

[0021] Furthermore, the concentration of resveratrol added was 4 g / L.

[0022] Furthermore, the fermentation temperature is 28-32℃, and the pH is 5.5-6.5.

[0023] Furthermore, the fermentation temperature was 30℃ and the pH was 6.0.

[0024] In summary, the present invention has the following beneficial effects: 1. By screening for glycosidase genes with specific functions, key catalytic elements for resveratrol synthesis were identified. Pichia pastoris GS115 was genetically engineered, and single or multiple glycosidase genes were knocked out to optimize its metabolic background, thus constructing a high-yielding resveratrol-producing chassis strain. A UDPG supply module was introduced into the chassis strain, and its supply capacity was enhanced through tandem or iterative integration, providing sufficient precursors for resveratrol synthesis.

[0025] 2. This invention, by knocking out endogenous β-glucosidase genes (891B, EXG1) and introducing a UDPG supply module (YNK1 / PGM2 / galU), resulted in recombinant strains (such as HZG13) that achieved a resveratrol yield of 1.86 g / L in shake-flask fermentation, approximately 2.6 times higher than the original strain HZG01 (0.5 g / L). After knocking out the key glycoside-degrading enzyme gene, strain TG14 maintained a stable resveratrol yield of 0.7 g / L within 48-120 h, while the original strain HZG01's yield decreased to 0.1 g / L after 168 h, effectively solving the problem of product degradation by endogenous enzymes.

[0026] 3. This invention screens and integrates UDPG supply module genes such as PGM2, YNK1, and galU, enhancing intracellular UDPG regeneration capacity and providing sufficient precursors for glycosylation reactions, thereby significantly improving the conversion efficiency of resveratrol to polysaccharide. Strong constitutive promoters such as pTDH3, pGAP, and pGCW14 are screened, which can stably and efficiently drive gene expression in different culture media. Two 2A peptides, OpbuCPV18 and EMCV, are screened, enabling efficient co-expression of multiple genes and simplifying the construction of metabolic pathways. Attached Figure Description

[0027] Figure 1 The figure shows the activity evaluation results of the promoter elements in the polygalactosidase biosynthetic pathway. Figure 2 The fluorescence intensity evaluation diagram for the synthesis of polygalactosidase based on the 2A peptide strategy is shown. Figure 3 The figure shows the construction and HPLC validation results of the polydextrin synthesis pathway in Komagataella phaffii GS115; Figure 4Figure showing the results of glycoside-degrading enzyme activity assay and candidate gene knockout validation. Figure 5 Precursor supply module screening and UDP-G steady-state regulation diagram; Figure 6 Figure showing the comparison of polygalactoside synthesis performance between modified strain TG14 and wild-type HZG01; Figure 7 Figure showing the comparison results of precursor supply performance of the modified strain TG14. Detailed Implementation

[0028] 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.

[0029] Example 1: Development of a Synthetic Biology Element Library 1. Construction and evaluation of the startup component library To improve the expression level of the strain, this study screened 21 promoters by fusing red fluorescent protein as a reporter molecule. The screened promoters were pPDC1, pRGI2, pSSA3, pADH2, pTEF1, pCDC1, pGAP, pGCW14, p0061, p0208, p0090, p0832, p0932, p0570, p0002, and p0082.

[0030] Fermentation validation was then performed. This study used BMTY, BMGY, and BMMY media for screening, and the results are as follows: Figure 1 As shown, Figure 1 In the table, A represents the expression intensity of different promoters in BMTY medium. HR (wild type) and GS115 (wild type) were used as controls; B represents the expression intensity of different promoters in BMGY medium; and C represents the expression intensity of different promoters in BMMY medium.

[0031] Depend on Figure 1 It was found that, in BMTY medium (A), with HR and GS115 wild-type strains as controls, the expression intensity of different promoters showed significant differences. Among them, the constitutive promoter pTDH3 had the highest fluorescence intensity, reaching approximately 3300 (Fluorescence / OD). 600 The second highest value was pGAP, which was approximately 2900 (Fluorescence / OD). 600 ) and pGCW14 are approximately 2500 (Fluorescence / OD) 600), while the expression intensity of promoter p0061 was the lowest, Fluorescence / OD 600 The fluorescence intensity was nearly zero, indicating that constitutive promoters can drive efficient protein expression even under non-inducible conditions. In BMGY medium (B), the overall fluorescence intensity level was generally lower than in BMTY medium, with the highest expression intensity for promoter p0586, approximately 1700 (Fluorescence / OD). 600 ), while pGAP is approximately 500 (Fluorescence / OD). 600 ) and pGCW14 is approximately 650 (Fluorescence / OD) 600 The expression intensity of pGCW14 was the second highest; in contrast, the expression intensity of promoter p0061 was also the lowest, suggesting that the transcriptional activity of some inducible promoters was significantly inhibited under glycerol-induced conditions. Under methanol-induced conditions in BMMY medium (C), the expression intensities of pGCW14 and p0932 were the most prominent, with the fluorescence intensity of pGCW14 reaching approximately 2600 (Fluorescence / OD). 600 p0061 was the promoter with the lowest expression intensity in all culture media, with a fluorescence intensity of only about 10 (Fluorescence / OD). 600 This further confirms the limitations of its transcriptional activity.

[0032] The experimental results from the three culture media showed that the promoter fluorescence intensity of the glycerol and methanol-induced systems was generally low, while the constitutive promoters pTDH3, pGAP, and pGCW14 exhibited stable and high expression intensities under different culture conditions. This indicates that using strong promoters to independently drive the expression of each gene can effectively regulate gene transcription levels, which is beneficial for balancing metabolic flux and avoiding metabolic burden or bottlenecks caused by single-gene overexpression. Therefore, this study ultimately selected pTDH3, pGAP, and pGCW14 as the core promoter elements for subsequent engineering modification of the polygalactosidase biosynthetic pathway.

[0033] 2. Screening of 2A peptide sequences To screen for 2A peptides suitable for Pichia pastoris GS115, this study selected seven 2A peptides that have been proven effective in Saccharomyces cerevisiae, including P2A, F2A, opbuCPV18, T2A, PnPV2A1, EMCV, and ERBV1, and constructed corresponding screening expression cassettes.

[0034] Fermentation validation was then performed, and BMTY, BMGY, and BMMY media were used for screening. Figure 2 As shown. Figure 2In the table, A represents the green fluorescence expression intensity of 2A peptide in BMTY medium, with GFP (wild type) as the control; B represents the green fluorescence expression intensity of 2A peptide in BMGY medium, with GFP (wild type) as the control; C represents the green fluorescence expression intensity of 2A peptide in BMMY medium, with GFP (wild type) as the control; D represents the red fluorescence expression intensity of 2A peptide in BMTY medium, with mcherry (wild type) as the control; E represents the red fluorescence expression intensity of 2A peptide in BMTY medium, with mcherry (wild type) as the control; and F represents the red fluorescence expression intensity of 2A peptide in BMMY medium, with mcherry (wild type) as the control.

[0035] Depend on Figure 2 It can be seen that in BMTY medium (A, D), the fluorescence intensity of GFP is generally maintained at approximately 15000–24000 Fluorescence / OD. 600 Among them, OpbuCPV18 and EMCV showed the highest GFP fluorescence intensity, both reaching approximately 24,000 Fluorescence / OD. 600 The corresponding mCherry fluorescence intensity is approximately 200–250 Fluorescence / OD. 600 Among them, EMCV exhibited the highest mCherry fluorescence intensity, approximately 1200 Fluorescence / OD. 600 In BMGY medium (B, E), the overall fluorescence intensity of GFP ranged from approximately 13,000 to 22,000 Fluorescence / OD. 600 The GFP fluorescence intensity of OpbuCPV18 and EMCV remained at a high level, approximately 18,000 and 17,000 Fluorescence / OD, respectively. 600 The corresponding mCherry fluorescence intensity is approximately 600–1200 Fluorescence / OD. 600 The mCherry fluorescence intensity of EMCV is approximately 1200 Fluorescence / OD. 600 The fluorescence intensity of GFP was significantly higher than that of other 2A peptides. In BMMY medium (C, F), the fluorescence intensity of GFP was mostly between approximately 8000–10000 fluorescence / OD. 600 ERBV1 exhibited the highest GFP fluorescence intensity, approximately 10500 Fluorescence / OD. 600 T2A exhibits the lowest GFP fluorescence intensity, at only approximately 1100 Fluorescence / OD. 600 The corresponding mCherry fluorescence intensity is approximately 400–1200 Fluorescence / OD. 600Among them, T2A had the highest mCherry fluorescence intensity, approximately 1200 Fluorescence / OD. 600 Based on the results from the three culture media, OpbuCPV18 and EMCV maintained high and stable levels of GFP and mCherry expression in both BMTY and BMGY media, while the fluorescence intensity of each 2A peptide was generally low in the methanol-induced system (BMMY). This indicates that OpbuCPV18 and EMCV are preferred 2A peptides for efficient multi-gene co-expression in Pichia pastoris and can be used for subsequent metabolic engineering of the polygalactosidase biosynthetic pathway.

[0036] Example 2 Biosynthesis and Identification of Polygonin 1. Construction of the polygalactoside synthesis gene Primers containing the homologous arm of the key gene R3GAT for polydipsia synthesis were designed to amplify the target gene fragment from the polydipsia genome. The recovered PCR product was ligated into the pBBR vector via homologous recombination and transformed into dH5α competent cells. Single clones were selected for colony PCR identification. Plasmids were extracted from the positive transformants and sequenced.

[0037] 2. Polygonum cuspidatum glycoside yield test Design of a CRISPR-Cas9 plasmid system: The Cas9 expression cassette was integrated into the AOX1 site, with sgRNA targeting the gene; the donor DNA was the tandem selection marker HIS4-HygB containing homologous arms, which was introduced into GS115 competent cells for expression via electroporation. Verification was performed using HygB anti-plate and colony PCR. After confirmation, fermentation was conducted to determine the yield of polygalactosidase. Seed culture: YPD, 30 ℃, 220 rpm, 24 h, OD... 600 ≈8; based on the initial OD 600 =0.20% inoculated into 30 mL BMTY medium; substrate: resveratrol 4 g / L added at once (purity ≥98%, sterilized by 0.22 μm filtration, dissolved in DMSO); parameters: 30 ℃, 220 rpm, pH 6.0; fermentation for 168 h, 1 mL sample taken every 24 h. Yeast cells were frozen at 20 ℃, and the yield of polygalactosidase and the remaining amount of resveratrol were determined by standardized HPLC. Yeast cell density was also measured. The results are as follows: Figure 3 As shown. Figure 3 In the figure, A shows the change in polygalactoside production of the HZG01 engineered strain with fermentation time, and B shows the HPLC chromatograms of resveratrol and polygalactoside standards (top 1.2) and HZG01 fermentation broth sample (bottom).

[0038] Depend on Figure 3It was found that the maximum titer of polygalactoside reached 0.5 g / L after 48 h of fermentation, and after 168 h, most of the polygalactoside had been degraded, with a yield of only 0.1 g / L. HPLC analysis (B) showed that the HZG01 fermentation broth sample exhibited a clear characteristic peak at the retention time corresponding to the polygalactoside standard, and a residual substrate signal was detected at the retention time of the resveratrol standard, directly confirming the successful formation of polygalactoside in the fermentation broth and reflecting the conversion process of exogenous resveratrol to the target product. Retention time 7.9 min was for polygalactoside, and 9.25 min was for resveratrol; the main product peak was consistent with the polygalactoside standard, confirming the successful synthesis of polygalactoside via this pathway.

[0039] Example 3 Screening of glycoside-degrading enzymes 1. Knockout yield test of glycoside-degrading enzyme gene (1) Discovery of glycosidases: Using the whole genome of Pichia pastoris GS115 as the object, the GH1, GH3, GH5 and GH30 families were scanned using the Hidden Markov Model (HMM), and a total of 11 candidate β-glucosidase genes were annotated. (2) Yield determination: using glucose as the carbon source (initial OD) 600 =0.2), 4 g / L of the substrate polydextrin (purity ≥98%, HPLC detection) was added, and the reaction was carried out at 30℃ and 220 rpm for 48 h. After 48 h of degradation reaction using polydextrin as the substrate, two strains (TG05 and TG09) with degradation activities exceeding 90% were identified by HPLC quantitative analysis. Their corresponding target genes were 891B (AT250_GQ6800891) and EXG1 (PAS chr2-1_0454). The results are as follows. Figure 4 As shown. Figure 4 In the figure, A represents the remaining amount of polygalactoside (relative value), and B represents the amount of resveratrol degradation.

[0040] Depend on Figure 4It was found that, compared with the wild-type control HR, the residual amount of resveratrol in the TG05 and TG09 knockout strains in A was significantly better than that in other tested strains, suggesting that the glycosidases encoded by these two genes are key functional genes for resveratrol degradation. In the parallel detection of resveratrol degradation (B), TG05 and TG09 also showed consistent degradation phenotypes, and the target gene corresponding to strain TG11, SPR1 (AT250_GQ6805053), also showed less resveratrol degradation. To further confirm the reliability of the core target genes, EXG1 (PAS chr2-1_0454) and SPR1 (AT250_GQ6805053) were subsequently iteratively knocked out based on TG05, and the strains were named TG12 and TG13. The fermentation results finally confirmed that strain TG05 provided a clear molecular target for the subsequent construction of the "zero degradation" chassis strain.

[0041] Example 4: UDPG Supply Module Screening 1. Screening of UDPG supply modules: Ten key genes, including PGM2 (phosphoglucosuric enzyme) and UGP1 (UDP-glucose pyrophosphorylase), were screened using a genome-scale metabolic model. Knock-in expression cassettes (integrated into the pGAP site and pTEF1 promoter) were constructed for each gene. Different precursor supply genes were screened and introduced into strain HZG01 along with the natural promoter and terminator. Using YPD as seed culture, the strain was activated at 30 °C and 220 rmp for 24 h and then inoculated into BMTY medium (initial OD). 600 = 0.2), pH 6.0, after 24 h of fermentation, 4 g / L resveratrol (purity ≥99%) was added exogenously, dissolved in DMSO, and reacted for 48 h. The product polydipsia glycoside and substrate resveratrol were detected by HPLC. It was found that the yield of polydipsia glycoside was significantly higher than that of HZG01 (wild type). Three UDP supply modules (YNK1 / PGM2 / galU) were obtained and named HZG03; HZG07; and HZG011. This laid the metabolic foundation for subsequent high-density fermentation. Precursor supply module screening and UDP-G homeostasis regulation are as follows: Figure 5 As shown. Figure 5 In the diagram, A represents the intracellular UDP-glucose (UDPG) regeneration pathway, and B represents the yield of polygalactoside under different combinations of precursor supply genes.

[0042] Depend on Figure 5 It is known that PGM converts G6P into G1P, and UGP uses UTP to further synthesize UDPG from G1P; NDK replenishes the UTP pool, providing a continuous glycosyl donor for R3GAT, thus achieving the efficient conversion of resveratrol to polysaccharide.

[0043] 2. Enhanced UDPG Supply Module The key gene 891B for glycoside degrading enzyme was knocked out in the engineered strain HZG01, and the strain was named TG14. The modified strain TG14 maintained a concentration of 0.7 g / L for 48-120 h, while the engineered strain HZG01 reached a maximum of 0.4 g / L, with a subsequent continuous decrease, indicating a significant improvement in the efficiency of the modified strain. The results are as follows: Figure 6 As shown. Figure 6 In the table, A represents the time series of resveratrol production; B represents the time series of resveratrol residue; and C represents the cell density of strains HZG01 and TG14 at 0, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours.

[0044] Based on the three key UDPG supply genes previously screened, they were overexpressed and integrated into TG14 cells for glutinin activity testing. Subsequently, the screened UDPG supply module genes (YNK1 / PGM2 / galU) were introduced into TG14 cells and named HZG12, HZG13, and HZG14, respectively, for plate fermentation verification. The results are as follows: Figure 7 As shown, Figure 7 In the diagram, A represents the yield of polygalactoside, B represents the residual amount of resveratrol, and C represents the cell density of strains TG14, HZG12, HZG13, and HZG14 after 48 hours of fermentation. Finally, the highest-yielding strain, HZG13, achieved a resveratrol yield of 1.86 g / L, a 2.6-fold increase compared to HZG01. In plate fermentation, the modified strain TG14 achieved a resveratrol yield of 1.45 g / L, while HZG13 achieved 1.7 g / L, indicating a significant improvement in the efficiency of the modified strains.

[0045] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a recombinant Pichia pastoris strain, characterized in that, Includes the following steps: A promoter and 2A peptide element library was constructed, the R3GAT gene was introduced into the Pichia pastoris genome, the endogenous β-glucosidase gene was discovered and knocked out, and then the UDP-glucose supply module gene was optimized and integrated to obtain a recombinant Pichia pastoris strain.

2. The preparation method according to claim 1, characterized in that, The Pichia pastoris is Pichia pastoris GS115.

3. The preparation method according to claim 1, characterized in that, The 2A peptide is OpbuCPV18 or EMCV.

4. The preparation method according to claim 1, characterized in that, The endogenous β-glucosidase gene is the 891B gene or / and the EXG1 gene.

5. The preparation method according to claim 1, characterized in that, The UDP-glucose supply module is at least one of the YNK1, PGM2 and galU genes, and the gene in the UDP-glucose supply module is expressed by a constitutive promoter, which is at least one of pTDH3, pGAP and pGCW14.

6. The recombinant Pichia pastoris strain prepared by the preparation method according to any one of claims 1-5.

7. The use of the recombinant Pichia pastoris strain according to claim 6 in the production of polygalactoside or food, health products, cosmetics or pharmaceuticals containing polygalactoside.

8. A method for producing polygalactoside, characterized in that, Includes the following steps: The recombinant Pichia pastoris strain and resveratrol described in claim 5 were added to the fermentation medium for fermentation, and polygalactoside was collected.

9. The method as described in claim 8, characterized in that, The fermentation medium is one of BMTY medium, BMGY medium and BMMY medium.

10. The method as described in claim 8, characterized in that, The concentration of resveratrol added is 2-6 g / L; the fermentation temperature is 28-32℃, and the pH is 5.5-6.5.

Images