Ethylene glycol high-tolerance pichia pastoris and application thereof
By constructing a Pichia cerevisia strain LETBE1022 that is resistant to high concentration of ethylene glycol and expressing PET hydrolase, the problem of difficult degradation of PET plastics is solved, and efficient biodegradation effect is achieved.
Patent Information
- Application Number
- CN202510297455.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art is difficult to effectively degrade polyethylene terephthalate (PET) plastics, resulting in environmental pollution and lacks efficient biodegradation methods.
A Pichia sacchariasis strain LETBE1022 that is resistant to high concentration of ethylene glycol was developed, and a PET hydrolase was constructed through genetic engineering. The yeast strain was used to produce PET hydrolase in a high concentration of ethylene glycol environment to achieve biodegradation of PET.
While maintaining good growth performance in a high concentration of ethylene glycol environment, the PET degradation efficiency is significantly improved and the efficient biodegradation of PET is achieved.
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Abstract
Description
Technical Field
[0001] The present invention relates to Pichia pastoris with high ethylene glycol tolerance and its application, belonging to the technical field of microbial engineering. Background Art
[0002] Polyethylene terephthalate (PET) is a kind of plastic. Due to its advantages such as high mechanical strength, low air permeability, light weight, and low cost, it has been widely used worldwide. A large amount of PET waste is difficult to degrade in the natural state, resulting in the accumulation of PET in the global ecological system, causing the loss of soil nutrients and soil compaction, and a large amount of plastics floating in the ocean will have an adverse impact on the marine ecosystem.
[0003] Plastics have a wide and important application in society, but the mismanagement of these fossil fuel-derived resources is causing widespread pollution. The global plastic waste crisis is now considered to be one of the most pressing environmental problems facing our planet, prompting an urgent call for new technologies to achieve a circular plastic economy. Biodegradation technology has gradually become a research hotspot in the field of plastic degradation due to its advantages such as green pollution-free and low cost. Polyethylene terephthalate (PET) is an abundant and very useful material with a wide range of applications in society. However, there is an urgent need to develop technologies to evaluate post-consumer PET waste in order to address plastic pollution and move towards a circular economy. Although PET degradation and recycling technologies have been reported, the examples focus on reusing the resulting monomers to produce more PET. The product ethylene glycol (EG) of PET degradation can be utilized by microorganisms as a carbon source for product expression or metabolic transformation to generate valuable compounds. It has been reported that many microorganisms, especially some bacteria and yeasts, can utilize EG as a carbon source for growth and metabolism. Pichia pastoris shows good growth ability in the metabolism of EG. Therefore, it is of great significance to use Pichia pastoris to express some PET-degrading enzymes using EG as a carbon source. Summary of the Invention
[0004] The present invention provides a strain of Pichia pastoris LETBE1022, classified and named Pichia pastoris LETBE1022, which was deposited at the China Center for Type Culture Collection on December 09, 2024, with the deposit number CCTCC NO: M 20242753, and the deposit address is Wuhan University, Wuhan, China.
[0005] The present invention provides the application of the Pichia pastoris LETBE1022 in constructing a genetically engineered bacterium as a chassis cell.
[0006] In one embodiment, the genetically engineered bacterium expresses a protein using the Pichia pastoris as a host.
[0007] In one embodiment, the protein includes but is not limited to PET hydrolase.
[0008] In one embodiment, the PET hydrolase includes but is not limited to BhrPETase, FastPETase, DuraPETase or 4Mz; the nucleotide sequence encoding BhrPETase is shown as SEQ ID NO.3; the nucleotide sequence encoding FastPETase is shown as SEQ ID NO.5; the nucleotide sequence encoding DuraPETase is shown as SEQ ID NO.6; the nucleotide sequence encoding 4Mz is shown as SEQ ID NO.7.
[0009] In one embodiment, the PET hydrolase is expressed using pPIC9K as a vector.
[0010] In one embodiment, using pPIC9K as a vector, the promoter P AOX1 regulates the expression of PET hydrolase.
[0011] In one embodiment, a signal peptide is further contained upstream of the PET hydrolase, and the sequence encoding the signal peptide is shown as SEQ ID NO.2.
[0012] The present invention also provides a recombinant Pichia pastoris producing PET hydrolase, which uses the Pichia pastoris LETBE1022 as a host to express PET hydrolase.
[0013] The present invention provides a microbial preparation containing the recombinant Pichia pastoris.
[0014] In one embodiment, the microbial preparation contains the recombinant Pichia pastoris live cells.
[0015] In one embodiment, the dosage form of the microbial preparation includes at least one of granules, liquid, and dry powder.
[0016] The present invention also provides a method for constructing the recombinant microbial cells, which is to transform the recombinant expression vector into the Pichia pastoris LETBE1022.
[0017] In one embodiment, the transformation method includes but is not limited to electroporation, calcium phosphate precipitation, heat shock, lithium chloride method, lithium acetate method, microinjection, lipofection, etc.
[0018] The present invention also provides a method for preparing a PET degrading enzyme, which is to culture the engineered bacteria in a liquid environment containing EG.
[0019] In one embodiment, the liquid environment containing EG includes but is not limited to PET degradation solution.
[0020] In one embodiment, the liquid environment containing EG includes but is not limited to M9 medium containing EG.
[0021] In one embodiment, the liquid environment containing EG is a mixture of PET degradation solution and M9 medium.
[0022] In one embodiment, the EG content in the liquid environment containing EG is ≥5 mg / mL.
[0023] In one embodiment, the EG content in the liquid environment containing EG is 5 - 140 mg / mL.
[0024] In one embodiment, the PET degradation solution is a solution containing EG after degradation of PET products (such as beverage bottles, polyester fibers, etc.).
[0025] In one embodiment, the PET degradation solution is a PET residual solution that has been degraded from PET products (such as beverage bottles, polyester fibers, etc.), with TPA removed and only containing EG.
[0026] In one embodiment, the cultivation is carried out at 30 °C for at least 24 h.
[0027] In one embodiment, the cultivation temperature is specifically 30 - 40 °C, or 30 - 35 °C, or 35 - 40 °C.
[0028] In one embodiment, the method is to inoculate the genetically engineered bacteria with an inoculation amount of ≥1% (v / v).
[0029] In one embodiment, the inoculation amount is calculated as the mass - volume percentage (wt%) of the wet cell weight of the bacteria to the solution volume, specifically 1 - 12%, or 2 - 12%, or 4 - 12%, or 4 - 6%, or 4 - 8%, or 4 - 10%.
[0030] In one embodiment, the cultivation time is at least 24 h.
[0031] In one embodiment, the cultivation is carried out for 24 - 96 h.
[0032] In one embodiment, the method is to cultivate the genetically engineered bacteria in a seed medium for 24 h, collect the bacterial cells, inoculate them into a reaction system containing EG or PET degradation solution, and control the pH at 7.0 - 8.0 and the temperature at 30 °C - 35 °C during the transformation process for 24 - 96 h.
[0033] The present invention also provides the use of the Pichia pastoris LETBE1022 or the engineered Pichia pastoris strain in the field of plastic degradation.
[0034] In one embodiment, the use includes, but is not limited to, preparing a PET hydrolase and using it for PET degradation.
[0035] In one embodiment, the use includes, but is not limited to, fermenting a PET hydrolysis solution to prepare a PET hydrolase.
[0036] Beneficial effects:
[0037] (1) Through ARTP mutagenesis, the present invention obtains a Pichia pastoris strain LETBE1022 that is resistant to high concentrations of EG. Compared with the wild-type strain, it can grow and reproduce in 8% EG without losing its original performance, and has strong antioxidant capacity and genetic stability. Placing it in a PET degradation solution to prepare a PET degrading enzyme or other recombinant proteins can effectively increase the utilization efficiency of the PET degradation solution.
[0038] (2) The present invention transforms a plasmid containing the BhrPETase synthetic expression unit of the PET hydrolase into the Pichia pastoris strain LETBE1022 that is resistant to high concentrations of EG, and the constructed engineered Pichia pastoris strain can produce a PET hydrolase using a PET degradation solution.
[0039] Biological material preservation
[0040] Pichia pastoris LETBE1022, classified as Pichia pastoris LETBE1022, was deposited at the China Center for Type Culture Collection on December 09, 2024, with the deposit number CCTCC NO: M20242753, and the deposit address is Wuhan University, Wuhan, China. Description of the drawings
[0041] Figure 1 For the genetic stability analysis of the mutagenized strain LETBE1022. Detailed implementation manners
[0042] As used herein, the terms "comprising", "including", "having", and "containing" mean "including but not limited to", "including but not limited to", "having but not limited to", "containing but not limited to", and may be used interchangeably with the corresponding phrases. Unless the context clearly indicates otherwise, the term "or" as used herein is used to mean the term "and / or" and may be used interchangeably therewith. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, the suitable methods and material examples set forth below are illustrative only and do not limit the present invention in any way.
[0043] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The present invention will be further described below in conjunction with specific examples for better understanding, but the protection scope of the present invention is not limited only to the following description.
[0044] (I) Reagents and Sources
[0045] The PCR enzymes involved in the following examples were purchased from BaoRi Medical Biotechnology Co., Ltd. The gene sequencing involved in the following examples was undertaken by Genewiz Biotechnology Co., Ltd. The ethylene glycol (EG) involved in the following examples was purchased from Sigma. The polyethylene terephthalate (PET) plastics, mono(2-hydroxyethyl) terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), and ethylene glycol (EG) involved in the following examples were purchased from Sigma.
[0046] (II) Medium Formulations:
[0047] YPD Liquid Medium (w / v): Peptone 2%, molecular grade yeast extract 1%, glucose 2%.
[0048] YPD Solid Medium: In 150 mL of LB liquid medium, add 2.5 to 2.8 g of agar powder.
[0049] Screening Medium: Peptone 2%, yeast extract 1%, EG 8%.
[0050] Fermentation Medium BMGY: Yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, add water to 895 mL, sterilize at 121 °C for 20 min, then after the temperature drops to 60 °C, add 100 mL of 10×YNB (13.4 g / L) and 1 mL of 500× biotin (4×10 -4g / L), 10 mL of glycerol.
[0051] Fermentation medium BMMY: 10 g / L of yeast extract, 20 g / L of peptone, 3 g / L of K2HPO4, 11.8 g / L of KH2PO4, add water to 895 mL, sterilize at 121 °C for 20 min, and then add 100 mL of 10×YNB (13.4 g / L), 1 mL of 500×biotin (4×10 -4 g / L), 5 mL of methanol on the laminar flow bench after the temperature drops to 60 °C.
[0052] (III) Detection method:
[0053] Determination of EG concentration:
[0054] Standard sample treatment: Weigh the standard sample of EG and dissolve it in dimethyl sulfoxide (DMSO) to make a stock solution. Dilute the stock solution with sterile water to a 0.1 mg / mL standard sample solution, filter it with a 0.22 μM filter head, and inject it into the liquid phase bottle with a syringe for HPLC detection. Plot the standard curve of EG with the EG concentration on the x-axis and the peak area on the y-axis.
[0055] Sample treatment: Let the culture solution stand for 10 min, take 5 mL of the supernatant, centrifuge at 12000 rpm for 8 min, filter it with a 0.22 μM filter head, inject it into the liquid phase bottle with a syringe, and perform HPLC detection. The specific detection method is as follows: Use an Agilent 1260 high-performance liquid chromatograph, a DAD detector, and an Agilent C18 chromatographic column (5 μm, 4.6 mm i.d.×150 mm); High-performance liquid chromatography conditions: Mobile phase: methanol: 0.5% glacial acetic acid (v / v) = 30:70; Flow rate 1.0 mL / min; Injection volume 10 μL, detection wavelength 280 nm, column temperature 30 °C.
[0056] Determination of PET degrading enzyme activity:
[0057] (1) Plotting the pNP standard curve
[0058] Weigh 0.0139 g of p-nitrophenol, dissolve it in 10 mM pH 8.0 Tris-HCl buffer to a volume of 1000 mL to prepare a 100 μmol / L p-nitrophenol stock solution, and then dilute it with Tris-HCl buffer to 0, 20, 40, 60, 80, 100 μmol / L. Measure the absorbance at a wavelength of 405 nm with a cuvette on a spectrophotometer. Plot the standard curve A = a*C + b with the p-nitrophenol concentration C on the x-axis and the absorbance A on the y-axis.
[0059] (2) Determination of PET degrading enzyme activity
[0060] Preparation of the substrate for enzyme activity assay: Accurately weigh 0.1046 g of p-nitrophenyl butyrate (pNPB, Sigma, CAS 2635-84-9), and make up the volume to 10 mL with acetonitrile (using a volumetric flask), to prepare a pNPB solution with a concentration of 50 mmol·L -1 and store it sealed at -20 °C.
[0061] Preparation of the enzyme solution to be measured: Centrifuge the fermentation broth at 8000 rpm for 10 min, take the supernatant and dilute it appropriately. The dilution factor should be controlled so that the A value is within the range of 0.2 - 0.8.
[0062] Enzyme activity of PET-degrading enzyme: Take 1.5 mL of Tris-HCl buffer (pre-incubated at 37 °C for 10 min) into a 0.5 cm glass cuvette, and zero the absorbance at a wavelength of 405 nm. Take 1.44 mL of pre-warmed 10 mM pH 8.0 Tris-HCl buffer into a quartz cuvette. Use a 50 μL micropipette to transfer 30 μL of the diluted sample to be measured and add it to the above quartz cuvette. Then use a 50 μL micropipette to transfer 30 μL of the substrate solution and add it to the above quartz cuvette. After mixing, immediately place it in a visible spectrophotometer and measure the A value at a wavelength of 405 nm. Record the A value every 5 seconds, and the reaction time is 1 minute. Definition of enzyme activity: The amount of enzyme required to catalyze the substrate to produce 1 μmol of pNP per unit time is defined as one enzyme activity unit.
[0063] Calculation:
[0064] Enzyme activity (U / mL) = [[(K - b) * V1] / [a * V2 * 1000]] × N;
[0065] Where: K: The slope of the curve formed by the A values measured at different times and time (min) during the enzyme reaction; V1: Reaction volume (mL); V2: Enzyme addition volume (mL); N: Dilution factor.
[0066] Determination of the cell concentration of the recombinant bacterium:
[0067] Measure the OD value of the bacterial solution at a wavelength of 600 nm using a spectrophotometer.
[0068] Example 1: Mutagenesis of Pichia pastoris using ARTP mutagenesis technology
[0069] (1) Grow Pichia pastoris GS115 in YPD medium at 30 °C until the mid-logarithmic growth phase. Take 1 mL of the seed solution into a 1.5 mL sterile centrifuge tube, centrifuge at 4000 r / min, remove the supernatant, add 1 mL of PBS solution, mix well, centrifuge, and repeat three times to dilute the cell suspension so that the OD 600 value is 0.8 - 1.0.
[0070] (2) Take 10 μL of the diluted bacterial solution and smear it evenly on a sterile stainless-steel slide. Place the slide in the ARTP mutagenesis system for mutagenesis treatment. ARTP mutagenesis conditions: radio frequency power 120 W, treatment distance 2 mm, carrier gas flow rate 10 standard liters per minute, treatment temperature at room temperature (20 - 40 °C), treatment times 0, 40, 60, 80, 120, and 140 s.
[0071] (3) Put the treated slide into an EP tube containing 1 mL of sterile physiological saline, shake well, and then dilute it to 10 -3 、10 -4 、10 -5 . Take 50 μL of each dilution and spread it evenly on a plate. Make three parallel samples for each gradient and count the colonies after culturing at 30 °C for 24 h.
[0072] (4) Make a lethality curve to determine the relationship between lethality and mutagenesis time. The calculation formula is: Lethality = (Number of colonies in the control group - Number of colonies in the mutagenesis treatment group) / Number of colonies in the control group × 100%.
[0073] Example 2: Screening of strains with high tolerance to EG
[0074] (1) Primary screening: Dispense the seed medium into 96-well deep plates, add 0.08 g of EG to each well, and make up to 1 mL with YPD liquid medium. Pick single colonies on the plate into the seed liquid wells, culture at 200 r / min and 30 °C for 48 h. Pick the top five strains with the highest OD values of the bacterial liquid at 24 h and 48 h, transfer them to shake flasks, and store them in glycerol tubes.
[0075] (2) Secondary screening: First transfer the 10 strains picked from the 96-well deep plates and the original strain to 50 mL of YPD liquid medium, culture at 200 r / min and 30 °C for 24 h. Pipette 50 μL of the bacterial liquid and add it to 50 mL of YPD liquid medium with a final concentration of 80 mg / mL EG, culture at 200 r / min and 30 °C for 48 h. Compare the OD values of the bacterial liquid and the EG residual amounts of the 10 strains to obtain the mutagenized Pichia pastoris strains with better tolerance to EG. The results are shown in Table 1. The mutagenized strain LETBE1022 has the best growth state, and the cell density OD 600 reaches 3.2, and the residual EG amount in the medium is only 10 mg / mL.
[0076] Table 1 Growth of mutagenized Pichia pastoris strains in medium containing EG
[0077] Strain <![CDATA[OD 600 > EG (mg / mL) Wild strain 0.5 78 Mutagenized strain LETBE104 0.9 60 Mutagenized strain LETBE107 0.8 72 Mutagenized strain LETBE1013 1.1 58 Mutagenized strain LETBE1017 1.0 63 Mutagenized strain LETBE1029 2.3 20 Mutagenized strain LETBE1022 3.2 10 Mutagenized strain LETBE1047 1.6 34 Mutagenized strain LETBE1057 1.4 49 Mutagenized strain LETBE1080 1.5 48 Mutagenized strain LETBE1097 1.7 38
[0078] Example 3: Study on the passage stability of the mutagenized strain LETBE1022
[0079] The EG highly resistant strain LETBE1022 obtained in Example 2 was inoculated into 50 mL of YPD liquid medium and cultured at 200 r / min and 30 °C for 24 h. 50 μL of the bacterial solution was taken and serially diluted to an appropriate concentration, and then spread on a 50 mL YPD solid medium plate containing 80 mg / mL EG. After culturing at 30 °C for 24 h, 1 single colony was randomly selected and inoculated on a 50 mL YPD solid medium plate containing 80 mg / mL EG, and cultured at 30 °C for 24 h (the 1st generation). 1 single colony of the bacteria from the above plate in the 1st generation was randomly selected and inoculated on a 50 mL YPD solid medium plate containing 80 mg / mL EG, and cultured at 30 °C for 24 h (the 2nd generation). The above steps were continuously transferred 30 times. 5 single colonies were selected from the bacteria in the 30th generation and inoculated into 50 mL of YPD liquid medium containing 80 mg / mL EG, and cultured at 200 r / min and 30 °C for 48 h. The OD values of the bacterial solutions were compared. The results are as Figure 1 shown. After the strain was passaged 30 times, there was no significant difference in the growth bacterial concentration compared with that of the 1st generation bacteria. After culturing for 48 h, the OD 600 of the bacterial concentration reached 3.1 - 3.3. This strain was deposited in the China Center for Type Culture Collection, and the deposit number was CCTCC NO: M 20242753.
[0080] Example 4: Growth status of the EG highly resistant strain LETBE1022 at different EG concentrations
[0081] The EG highly resistant strain LETBE1022 obtained in Example 2 was transferred into 50 mL of YPD liquid medium and cultured at 200 r / min and 30 °C for 24 h. 50 μL of the bacterial solution was respectively added to YPD liquid media containing different final concentrations of EG, and cultured at 200 r / min and 30 °C for 48 h. The OD values of the bacterial solutions and the residual amount of EG in the fermentation broth were compared. The results are shown in Table 2. With the increase in the EG concentration, the growth rate of the wild strain decreased significantly; when the EG concentration reached 100 mg / mL, the growth of the wild strain was completely inhibited. When the EG concentration was 5 - 100 mg / mL, the mutagenized strain LETBE1022 grew normally and showed good tolerance activity to EG. When the EG concentration was 120 - 140 mg / mL, the growth of the mutagenized strain LETBE1022 was inhibited to a certain extent.
[0082] Table 2 Effects of different concentrations of EG on the growth of wild and mutagenized strains
[0083]
[0084] Example 5: Preparation of competent cells of Pichia pastoris
[0085] The EG highly resistant strain LETBE1022 obtained in Example 2 was inoculated into 10 mL of YPD medium and cultured at 30 °C and 200 r / min for 24 h. Then, 100 μL of the bacterial solution was taken and inoculated into 100 mL of YPD medium, and cultured under the same conditions for 16 h until the OD of the bacteria 600 reached 1.5; all the bacteria were transferred into a sterilized and pre-cooled 50 mL centrifuge tube in a laminar flow hood, centrifuged at 4 °C and 4500 r / min for 5 min, the supernatant was discarded, and the bacteria were retained; 15 mL of sterile water was added to the above bacteria, and they were pipetted to resuspend, centrifuged at 4 °C and 4500 r / min for 5 min, the supernatant was discarded, and the bacteria were retained. This step was repeated once; 6 mL of pre-cooled sorbitol, 1 mL of Tris-HCl, 1 mL of LiAc, and 1 mL of sterile water were added to the centrifuged bacteria, pipetted and mixed evenly, 1 mL of sterilized dithiothreitol (DTT) was added, shaken well, and left to stand at room temperature for 20 min; centrifuged at 4 °C and 4500 r / min for 5 min, the supernatant was discarded, and the bacteria were retained; 10 mL of pre-cooled sorbitol was added to the above bacteria, and they were pipetted to resuspend, centrifuged at 4 °C and 4500 r / min for 5 min, the supernatant was discarded, and the bacteria were retained. This step was repeated twice; the bacteria were pipetted and resuspended with 1 mL of pre-cooled 1 mol / L sorbitol, and aliquoted into 1.5 mL EP tubes, 80 μL in each tube, and used immediately as prepared.
[0086] Example 6: Construction and verification of a Pichia pastoris engineering bacterium producing PET-degrading enzyme
[0087] Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to synthesize the pPIC9K-BhrPETase expression cassette. The plasmid was based on the pPIC9K plasmid backbone, and the expression cassette contained the inducible promoter P AOX1 + BhrPETase signal peptide + BhrPETase gene + protein translation terminator Teminator.
[0088] The specific structure description is as follows:
[0089] (1) Inducible promoter P AOX1 : The nucleotide sequence is as shown in SEQ ID NO.1;
[0090] (2) BhrPETase signal peptide: The nucleotide sequence is as shown in SEQ ID NO.2;
[0091] (3) BhrPETase gene: The gene is derived from Bacterium HR 29, and the nucleotide sequence is as shown in SEQ ID NO.3;
[0092] (4) Protein translation terminator Teminator: AOX1 - Teminator, the nucleotide sequence is shown in SEQ ID NO.4.
[0093] Furthermore, the synthesized recombinant plasmid pPIC9K - BhrPETase expression cassette was introduced into the competent cells of Pichia pastoris LETBE1022 by electroporation. After screening with the histidine selection marker, verification by colony PCR, and sequencing compliance, the Pichia pastoris engineering strain LETBE1022 - pPIC9K - BhrPETase was obtained. The engineering strain GS115 - pPIC9K - BhrPETase constructed with the wild strain as the host was used as the control strain.
[0094] The constructed Pichia pastoris engineering strain was inoculated into YPD liquid seed medium and cultured at 30 °C for 24 h. It was inoculated into 50 mL of BMGY liquid medium at 1% (w / w) and cultured at 30 °C for 24 h until the cell density OD 600 = 2 - 6; centrifuged at 4000 rpm for 10 min, the cells were collected and washed twice with ddH2O, and inoculated into YPD liquid medium containing 100 mg / mL EG at an inoculation amount of 2 wt%. During the fermentation process, the pH was controlled at 7.0, and fermentation was carried out at 200 r / min and 35 °C for 96 h. Methanol with a final concentration of 0.5% was added to the culture medium every 2 h for induction culture. After fermentation, the supernatant was taken by centrifugation at 8000 rpm and 4 °C for 10 min for enzyme activity determination. The results are shown in Table 3. When the EG concentration was 100 mg / mL, the engineering strain LETBE1022 - pPIC9K - BhrPETase neither grew nor produced enzymes. At an EG concentration of 100 mg / mL, the enzyme production of the engineering strain LETBE1022 - pPIC9K - BhrPETase increased with the prolongation of the culture time. At 96 h, the enzyme production of the engineering strain LETBE1022 - pPIC9K - BhrPETase was the highest, reaching 456.2 U / mL.
[0095] Table 3 Effects of Pichia pastoris enzyme production
[0096]
[0097]
[0098] Example 7: Effects of different EG concentrations on the production of PET - degrading enzymes by Pichia pastoris engineering bacteria
[0099] The engineered strain LETBE1022-pPIC9K-BhrPETase constructed in Example 6 was inoculated into YPD liquid seed medium and cultured at 30 °C for 24 h. It was inoculated into 50 mL of BMGY liquid medium at 1% (w / w) and cultured at 30 °C for 24 h until the cell density OD 600 = 2 - 6; centrifuged at 4000 rpm for 10 min, the cells were collected and washed twice with ddH2O, and inoculated into YPD liquid medium containing different concentrations of EG at an inoculation amount of 2 wt%. The pH was controlled at 7.0 during the fermentation process, and the fermentation was carried out at 200 r / min and 35 °C for 96 h. Methanol with a final concentration of 0.5% (v / v) was added to the culture medium every 2 h for induction culture. After the fermentation was completed, the supernatant was taken by centrifugation at 8000 rpm and 4 °C for 10 min for enzyme activity determination. The results are shown in Table 4. When the EG concentration reached 5 - 60 mg / mL, the engineered strain LETBE1022-pPIC9K-BhrPETase could utilize EG for growth and produce enzymes. When the EG concentration exceeded 60 mg / mL, the growth of the engineered strain LETBE1022-pPIC9K-BhrPETase was significantly inhibited. The enzyme production of the engineered strain LETBE1022-pPIC9K-BhrPETase increased with the increase of the EG concentration. When the EG concentration reached 100 mg / mL, the enzyme production of the engineered strain LETBE1022-pPIC9K-BhrPETase was the highest, reaching 456.2 U / mL.
[0100] Table 4 Effects of different EG concentrations on the production of degradation enzymes by Pichia pastoris engineered strains
[0101]
[0102]
[0103] Example 8: Effects of different temperatures on the production of PET-degrading enzymes by Pichia pastoris engineered strains
[0104] The engineered strain LETBE1022-pPIC9K-BhrPETase constructed in Example 6 was inoculated into YPD liquid seed medium and cultured at 30 °C for 24 h. It was inoculated into 50 mL of BMGY liquid medium at 1% (w / w) and cultured at 30 °C for 24 h until the cell density OD 600= 2 - 6; Centrifuge at 4000 rpm for 10 min, collect the cells and wash them twice with ddH2O, and inoculate them into YPD liquid medium containing 100 mg / mL EG at an inoculation amount of 2 wt%. During the fermentation process, the pH was controlled at 7.0, and the fermentation was carried out at 200 r / min at different temperatures (15 °C, 25 °C, 30 °C, 35 °C, 40 °C) for 96 h. Methanol with a final concentration of 0.5% (v / v) was added to the culture medium every 2 h for induction culture. After the fermentation was completed, the fermentation broth was centrifuged at 8000 r / min and 4 °C for 10 min, and the supernatant was taken for enzyme activity determination. The results are shown in Table 5. Temperature can significantly affect the enzyme-producing ability of the engineered strain LETBE1022-pPIC9K-BhrPETase. At 35 °C, the enzyme production of the engineered strain LETBE1022-pPIC9K-BhrPETase was the highest, reaching 564.2 U / mL.
[0105] Table 5 Effects of different temperatures on the production of PET-degrading enzymes by engineered Pichia pastoris
[0106] Temperature (°C) <![CDATA[OD 600 > Enzyme activity (U / mL) 15 2.1 121.1 25 3.8 265.4 30 4.6 456.2 35 5.2 564.8 40 4.2 415.4
[0107] Example 9: Effects of different inoculation amounts on the production of PET-degrading enzymes by engineered Pichia pastoris
[0108] Inoculate the engineered strain LETBE1022-pPIC9K-BhrPETase constructed in Example 6 into YPD liquid seed medium and culture it at 30 °C for 24 h. Inoculate it into 50 mL of BMGY liquid medium at 1% (w / w) and culture it at 30 °C for 24 h until the cell density OD 600 = 2 - 6; Centrifuge at 4000 rpm for 10 min, collect the cells and wash them twice with ddH2O, and inoculate them into YPD liquid medium containing 100 mg / mL EG at different inoculation amounts (1 wt%, 2 wt%, 4 wt%, 6 wt%, 8 wt%, 10 wt%, 12 wt%). During the fermentation process, the pH was controlled at 7.0, and the fermentation was carried out at 200 r / min and 35 °C for 96 h. After the fermentation was completed, centrifuge at 8000 r / min and 4 °C for 10 min and take the supernatant for enzyme activity determination. The results are shown in Table 6. As the inoculation amount increased, the density of the recombinant bacteria and the activity of the degrading enzyme gradually increased and then remained unchanged. When the inoculation amount was 4 wt%, the activity of the degrading enzyme could reach 597.9 U / mL.
[0109] Table 6 Effects of different inoculation amounts on the production of PET-degrading enzymes by engineered Pichia pastoris
[0110]
[0111] Example 10: Testing of different PET-degrading enzyme expression cassettes introduced into engineered Pichia pastoris
[0112] Construct the expression cassettes of different PET-degrading enzymes "P AOX1 -signal peptide-PET hydrolase-terminator" according to the method of Example 6, except that the BhrPETase gene in the recombinant plasmid pPIC9K-BhrPETase in Example 6 is replaced with the FastPETase gene (nucleotide sequence shown in SEQ ID NO.5), DuraPETase gene (nucleotide sequence shown in SEQ ID NO.6), and 4Mz gene (nucleotide sequence shown in SEQ ID NO.7) respectively to obtain recombinant plasmids pPIC9K-BhrPETase-FastPETase, pPIC9K-BhrPETase-DuraPETase, and pPIC9K-BhrPETase-4Mz.
[0113] Introduce the above recombinant plasmids into the mutant strain LETBE1022 by the transformation method in Example 5 to obtain engineered strains LETBE1022-pPIC9K-FastPETase, LETBE1022-pPIC9K-DuraPETase, and LETBE1022-pPIC9K-4Mz. The above engineered strains are inoculated into YPD liquid seed medium and cultured at 30 °C for 24 h. Inoculate at 1% (w / w) into 50 mL of BMGY liquid medium and culture at 30 °C for 24 h until the cell density OD 600 = 2 - 6; centrifuge at 4000 rpm for 10 min, collect the cells and wash them twice with ddH2O, and inoculate at an inoculation amount of 4 wt% into YPD liquid medium containing 100 mg / mL EG. During the fermentation process, the pH is controlled at 7.0, and fermentation is carried out at 200 r / min and 35 °C for 96 h. Methanol with a final concentration of 0.5% is added to the culture solution every 2 h for induction culture. After fermentation, centrifuge at 8000 r / min and 4 °C for 10 min, and take the supernatant for enzyme activity determination. The results are shown in Table 7. The engineered strains can produce PET-degrading enzymes using PET hydrolysis solution. The recombinant strains produce 623.9 U / mL of FastPETase, 697.6 U / mL of DuraPETase, and 721.5 U / mL of 4Mz.
[0114] Table 7 Production of PET-degrading enzymes by engineered Pichia pastoris
[0115]
[0116] Example 11: Preparation of PET hydrolysis solution
[0117] Referring to the previous research results of the inventor team, the enzyme protein of PET hydrolase (4Mz) was prepared according to the method in the paper "Directional-path modification strategy enhances PET hydrolase catalysis of plastic degradation". Add 1 L of 100 mM potassium phosphate buffer at pH 8, 200 g of PET powder (sieved through 500 mesh) and 0.2 g of 4Mz enzyme protein into a 3 L reactor, and react at 60 °C and 200 rpm for 96 h. During the reaction, maintain the pH value at 8 with 6 M NaOH. Filter the reaction solution to remove impurities, obtain the PET degradation solution and dilute it with 100 mM potassium phosphate buffer at pH 8 to obtain PET degradation solutions with different dilution factors (Table 8).
[0118] Table 8 PET degradation solutions with different dilution factors
[0119]
[0120]
[0121] Example 12: Preparation of residual PET degradation solution after removing TPA
[0122] Obtain the PET degradation solution by referring to the method described in Example 11. Add 6 M NaOH solution to 1 L of the PET degradation solution to adjust the pH of the PET degradation solution system to 9.0, so that TPA in the PET degradation solution dissolves in the alkaline solution, and then filter to remove impurities to obtain the supernatant. Then, slowly add 6 M HCl solution to the supernatant and gradually adjust it to pH = 4.0 to precipitate TPA again in the acidic environment, and let it stand overnight. Centrifuge and filter, discard the precipitate, and adjust the pH of the supernatant back to 7.0 to obtain the residual PET degradation solution containing only EG. Then dilute it with 100 mM potassium phosphate buffer at pH 8 to obtain residual PET degradation solutions with different dilution factors (Table 9).
[0123] Table 9 Residual PET degradation solutions with different dilution factors
[0124]
[0125] Example 13: Production of PET hydrolase by Pichia pastoris engineered bacteria using PET degradation solution
[0126] Inoculate the engineered bacteria LETBE1022-pPIC9K-BhrPETase constructed in Example 6 into the YPD liquid seed medium and culture it at 30 °C for 24 h. Inoculate it into 50 mL of BMGY liquid medium at 1% (w / w) and culture it at 30 °C for 24 h until the cell density OD 600= 2 - 6; Centrifuge at 4000 rpm for 10 min, wash twice with ddH2O, and inoculate into the PET degradation solution prepared in Example 7 or the residual PET degradation solution prepared in Example 11 at an inoculation amount of 2 wt% respectively. During the fermentation process, the pH is controlled at 7.0, and the fermentation is carried out at 200 r / min and 35 °C for 96 h. Methanol with a final concentration of 0.5% is added to the culture solution every 2 h for induced culture. After the fermentation is completed, centrifuge at 8000 r / min and 4 °C for 10 min, and take the supernatant for enzyme activity determination. The results are shown in Table 10. The engineered strain LETBE1022-pPIC9K-BhrPETase can use both the PET hydrolysis solution and the residual PET hydrolysis solution after removing TPA for the production of BhrPETase. When using the PET hydrolysis solution as the raw material, the degradation enzyme activity of the recombinant strain in the ×10 PET hydrolysis solution can reach 373.8 U / mL. When using the residual PET hydrolysis solution as the raw material, the degradation enzyme activity of the recombinant strain in the ×10 residual PET hydrolysis solution can reach 364.9 U / mL.
[0127] Table 10 Determination results of PET degrading enzymes in the fermentation broth
[0128]
[0129]
[0130] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims
1. Pichia pastoris LETBE1022 was deposited at the China Center for Type Culture Collection on December 09, 2024, with the deposit number CCTCC NO: M 20242753.
2. Use of the Pichia pastoris LETBE1022 according to claim 1 in constructing a genetically engineered bacterium as a chassis cell.
3. The application according to claim 2, wherein Expressing a protein using the Pichia pastoris LETBE1022 as a host; the protein includes but is not limited to PET hydrolase.
4. The application according to claim 3, characterized in that, The PET hydrolase includes but is not limited to BhrPETase, FastPETase, DuraPETase or 4Mz; the nucleotide sequence encoding BhrPETase is shown as SEQ ID NO.3; the nucleotide sequence encoding FastPETase is shown as SEQ ID NO.5; the nucleotide sequence encoding DuraPETase is shown as SEQ ID NO.6; the nucleotide sequence encoding 4Mz is shown as SEQ ID NO.
7.
5. Pichia pastoris engineering bacteria producing PET hydrolase, characterized in that, Using the Pichia pastoris LETBE1022 according to claim 1 as a host and pPIC9K as a vector to express PET hydrolase.
6. The Pichia pastoris engineering bacterium according to claim 5, wherein Regulate the expression of PET hydrolase with promoter P AOX1 Regulate the expression of PET hydrolase.
7. The engineered Pichia pastoris strain according to claim 5 or 6, characterized in that, The upstream of the PET hydrolase also contains a signal peptide, and the sequence encoding the signal peptide is shown as SEQ ID NO.
2.
8. A microbial preparation containing the Pichia pastoris engineered bacterium according to any one of claims 5 to 7.
9. A method for preparing a PET degrading enzyme, characterized in that, Culturing the Pichia pastoris engineered bacterium according to any one of claims 5 to 7 in a liquid environment containing EG.
10. Use of the Pichia pastoris LETBE1022 according to claim 1, or the Pichia pastoris engineered bacterium according to any one of claims 5 to 7, or the microbial preparation according to claim 8, or the method according to claim 9 in the field of plastic degradation.