A recombinant Pichia pastoris and its application in the production of Brazil gluten.

Abstract

This invention relates to a recombinant Pichia pastoris strain and its application in the production of carbapenem, belonging to the field of fermentation technology. This invention discovered that during the fermentation of carbapenem, some carbapenem accumulates and degrades within vacuoles, leading to a reduction in the extracellular secretion of carbapenem and ultimately resulting in low carbapenem yield in the fermentation broth. Through investigation, this invention found that knocking out the protease sequence shown in SEQ ID NO.2 or SEQ ID NO.3 can increase the extracellular secretion of carbapenem. Further optimization of relevant process parameters during fermentation resulted in a carbapenem yield of 4.01 g / L in a 3L fermenter, achieving high-yield carbapenem production.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a recombinant Pichia pastoris and its application in the production of Brazil gluten. Background Technology

[0002] Brazzein is a natural sweet protein extracted from the fruit of a wild plant in West Africa. This protein consists of 54 amino acid residues forming a single-chain polypeptide structure, including eight cysteine ​​residues that can form four pairs of intramolecular disulfide bonds, helping to maintain its stable three-dimensional conformation. Brazzein has a relative molecular mass of approximately 6500, an isoelectric point of 5, and a sweetness up to 2000 times that of an equal mass of sucrose. Among known sweet proteins, brazzein has the smallest molecular weight, the best water solubility, and exhibits excellent thermal and pH stability—its sweetness is retained even after heating at 80°C for 4 hours.

[0003] Pichia pastoris ( PiChia pastoris Pichia pastoris is a yeast capable of growing using methanol as its sole carbon and energy source. Its genome was sequenced in 2009, and it is currently widely used in recombinant protein expression and metabolic engineering research. As a eukaryotic expression system, Pichia pastoris offers advantages such as simple genetic manipulation, eukaryotic post-translational modification capabilities, high-density fermentation in inorganic salt media, and efficient secretion of recombinant proteins. Furthermore, this strain has been designated GRAS (Generally Recognized As Safe) by the U.S. Food and Drug Administration (FDA), making it suitable for use in the food and pharmaceutical industries.

[0004] When producing carbapenem using microbial fermentation, problems arise due to misfolding or folding within cells, resulting in secreted carbapenem lacking the correct protein structure and thus failing to perform its function, leading to a decrease or even loss of sweetness. Furthermore, as a sweetener, carbapenem production requires high purity, and existing technologies may suffer from high losses and low purity during purification. Moreover, carbapenem production technology based on Pichia pastoris suffers from technical drawbacks such as unstable expression during scale-up, product degradation, and high levels of impurities. Therefore, a new method for carbapenem production is urgently needed to address these issues. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problems of unstable expression and low yield of Brazilian sweet protein in the prior art.

[0006] To address the aforementioned technical problems, this invention provides a recombinant Pichia pastoris strain and its application in the production of carbapenem. This invention discovered that during the fermentation of carbapenem, some carbapenem aggregates and degrades within vacuoles, leading to a reduction in the extracellular secretion of carbapenem and ultimately resulting in low carbapenem yield in the fermentation broth. Through investigation, this invention found that knocking out the amino acid sequence of the protease, as shown in SEQ ID NO.2 or SEQ ID NO.3, can increase the extracellular secretion of carbapenem. Further optimization of relevant process parameters during fermentation resulted in a carbapenem yield of 4.01 g / L in a 3L fermenter, achieving high-yield carbapenem production.

[0007] The first objective of this invention is to provide a recombinant Pichia pastoris strain that heterologously expresses the gene sequence of Brazilin as shown in SEQ ID NO.1 and knocks out the amino acid sequence of the protease as shown in SEQ ID NO.2 or SEQ ID NO.3.

[0008] Furthermore, SEQ ID NO.1:

[0009] CAGGACAAGTGTAAGAAGGTGTACGAAAATTACCCCGTCAGTAAGTGCCAGCTCGCCAATCAATGTAACTATGATTGCAAACTAGATAAACACGCACGAAGCGGCGAGTGTTTCTATGATGAAAAAAGGAACTTACAATGCATTTGCGACTACTGTGAGTAT.

[0010] Furthermore, SEQ ID NO.2:

[0011] MKVGPSFFLGNIGVFGPRVMASKPFLSPAFNNAFKRITVQSSSLVTKIPKINLRNFATFNQLRMSGSSWSRGSNFSNLKTAALFSLVFCVGTTFATPYLMKYTPLAIFNKNPSLLVYSLIGINAAVFALWKAPQYWRVLSRYGLLEKDARFNKWSM IGSAFSHQDFWHIGMNMLALYSFGTTVASYVGASNFLIMYLNGAVLSSLASLAYPVLAGVSSMGASLGASGALFAILGSFSYLFPYAKILLFVFPIPGGAWIAFLASIGWNVCGCVFRWGSFDYAAHLGGSLVGIFYGWLIDQRRKEFKKNTRTVW.

[0012] Furthermore, SEQ ID NO.3:

[0013] .

[0014] Furthermore, the expression of the Brazilian sweet protein was initiated using the AOX1 promoter.

[0015] Furthermore, the Pichia pastoris strain is Pichia pastoris X33.

[0016] A second objective of this invention is to provide a microbial agent comprising the above-mentioned recombinant Pichia pastoris.

[0017] A third objective of this invention is to provide the application of the above-mentioned recombinant Pichia pastoris or the above-mentioned microbial agent in the production of carbapenem.

[0018] The fourth objective of this invention is to provide a method for high-yield Brazil protein, which involves inoculating the above-mentioned Pichia pastoris or the above-mentioned microbial agent into a fermentation medium for fermentation and culture, and inducing Brazil protein expression using methanol.

[0019] Furthermore, the fermentation medium comprises CaSO4, K2SO4, MgSO4·7H2O, KOH, glycerol, H3PO4, and YNB. ​​YNB is an amino-free yeast nitrogen source medium; the YNB used in this invention was purchased from Solarbio, product number Y8040.

[0020] Furthermore, the fermentation temperature was 30-31℃ during the first 48 hours of fermentation, and 27-28℃ during the 48th-146th hours of fermentation.

[0021] Furthermore, after fermentation has proceeded for 18-24 hours, glycerol and methanol are fed in at a volume ratio of (6-7):(3-4).

[0022] Furthermore, when the bacterial wet weight reaches 180g / L-220g / L, glycerol feeding is stopped. 1-2 hours after stopping glycerol feeding, methanol is added for induction.

[0023] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0024] This invention knocks out proteases in Pichia pastoris, screening and constructing effective protease knockout strains BM3 and BM8. The BM3 strain produced 118.6 mg / L of brassinolide, and the BM8 strain produced 319.2 mg / L, providing a good solution for the intracellular degradation of brassinolide. Furthermore, through systematic optimization of fermenter temperature, carbon source supply, and basal culture medium formulation, the BM8 strain achieved a brassinolide yield of 4.01 g / L in a 3L fermenter, realizing stable and high-yield expression of brassinolide. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0026] Figure 1 These are SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) images of Brazil protein after shake-flask fermentation, where 1-3 are electrophoresis patterns from three parallel experiments.

[0027] Figure 2 These are fermentation results of strains GM1-GM9 and BM1-BM9;

[0028] Figure 3 This is a data graph of a 3L fermenter after temperature optimization, where OD 600 This indicates the absorbance value at a wavelength of 600 nm.

[0029] Figure 4 This is a data graph of a 3L fermenter with optimized culture medium and carbon source;

[0030] Figure 5 This is a graph showing the fermentation data of the strain after the protease combination knockout in Comparative Example 1.

[0031] Figure 6 This is a graph showing the data from conventional fermentation in a 3L tank, as shown in Comparative Example 2. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0033] The detection methods and instruments used in the following embodiments:

[0034] (a) Detection of fluorescent proteins: ELISA reader.

[0035] (b) Diquinoline carboxylic acid (BCA) method: BCA kit (Beyotime Biotechnology), microplate reader, 37℃ incubator.

[0036] The culture medium components involved in the following examples are shown in Tables 1 and 2.

[0037] Table 1. Formulation of Pichia pastoris trace element 1 (PTM1) trace salt solution

[0038]

[0039] Table 2 Culture medium formulation

[0040]

[0041] (10×YNB was obtained by bringing 13.4g of YNB powder to a final volume of 100mL of sterile water.)

[0042] Example 1: Construction of a recombinant Pichia pastoris strain capable of secreting brassinolide

[0043] Based on the protein sequence of brassin published on the UniProt website (UniProt ID: P56552), codons were optimized according to the codon preference of Pichia pastoris, and the whole-genome plasmid was synthesized. The pPICZα-Brazzein plasmid was constructed using the primers shown in Table 3. To detect whether brassin is secreted extracellularly, the pPICZα-Brazzein plasmid was linearized by enzyme digestion and electroporated into Pichia pastoris X33 competent cells. After culturing for 2 hours, the cells were plated on yeast extract-tryptone-glucose medium (YPD medium) supplemented with bleomycin and cultured at 30°C until single colonies appeared. Single colonies were picked and colony polymerase chain reaction (PCR) was performed for verification. Positive single colonies were transferred to 2 mL of YPD medium supplemented with bleomycin and cultured overnight to obtain recombinant Pichia pastoris B seed culture.

[0044] Two mL of recombinant Pichia pastoris B seed culture was inoculated into a 250 mL Erlenmeyer flask containing 25 mL of methanol-buffered basal medium (BMMY medium). After culturing at 30°C and 220 rpm for 24 hours, the fermentation broth was centrifuged (4000×g, 5 min), the supernatant was discarded, and the cells were resuspended in 10 mL of sterile water. The mixture was then centrifuged again (4000×g, 5 min), the supernatant was discarded, and 25 mL of glycerol-buffered basal medium (BMGY medium) was added to resuspend the cells. The mixture was then transferred to a 250 mL Erlenmeyer flask and induced with methanol at 30°C and 220 rpm for 5 days, with 1% (v / v) methanol added every 24 hours.

[0045] Centrifuge the fermentation broth (4000×g, 5 minutes), collect the supernatant, and perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm whether the band position is brassinoprotein (results are shown below). Figure 1 (As shown). The content of brassinolide in the fermentation broth was 61.3 mg / L, determined by the quinoline carboxylic acid (BCA) method.

[0046] Table 3 Primers involved in Example 1

[0047]

[0048] Example 2: Protease screening and construction of knockout strains

[0049] To determine whether brassinosteroids accumulate in cells, the pPICZα-Brazzein-GFP plasmid was constructed using the primers shown in Table 4. After linearization by enzyme digestion, the pPICZα-Brazzein-GFP plasmid was electroporated into Pichia pastoris X33 competent cells. After culturing for 2 hours, the cells were plated on YPD medium plates supplemented with bleomycin and cultured at 30°C until single colonies appeared. Single colonies were picked for colony PCR. Positive single colonies were transferred to 2 mL of YPD (with bleomycin) and cultured overnight to obtain the seed culture of recombinant Pichia pastoris GB.

[0050] Table 4 Primers used for constructing the pPICZα-Brazzein-GFP plasmid

[0051]

[0052] 2 mL of recombinant Pichia pastoris GB seed culture was inoculated into a 250 mL Erlenmeyer flask containing 25 mL of BMMY medium. After culturing at 30°C and 220 rpm for 24 hours, the fermentation broth was centrifuged (4000×g, 5 min), the supernatant was discarded, and the cells were resuspended in 10 mL of sterile water. The cells were then centrifuged again (4000×g, 5 min), the supernatant was discarded, and the cells were resuspended in 25 mL of BMMY medium. The cells were then transferred to a 250 mL Erlenmeyer flask and induced to culture in methanol at 30°C and 220 rpm for 5 days, with 1% (v / v) methanol added every 24 hours.

[0053] Take 1 mL of fermentation broth, add 200 μL of fermentation broth to a 96-well plate, centrifuge the remaining fermentation broth, and add 200 μL of the supernatant to the 96-well plate. Detect fluorescence using a microplate reader. Excitation wavelength for green fluorescent protein (GFP) is 488 nm, emission wavelength is 523 nm, and gain is 50. Results are as follows: Figure 2 As shown in Figure A, brassinolide was found to accumulate intracellularly. After preparing the fermentation broth into a slide and observing it under a fluorescence microscope, brassinolide aggregation was observed within the vacuoles. The results are as follows: Figure 2 In the B group, it is considered that some of the brassinoprotein enters the degradation pathway.

[0054] Data from the Pichia pastoris genome were screened using "protease" as the keyword. Combined with the results of a basic local similarity search (BLAST) of related proteases on the uniprot website, nine proteases (M1-M9, amino acid sequences are shown in Table 5, * indicates stop codon) were selected. The gene positions related to each protease were located on the Pichia pastoris genome, and protease knockout plasmids (m1-m9) were constructed based on the upstream and downstream genes of the gene sites.

[0055] Table 5. Relevant amino acid sequences of M1-M9 proteases

[0056]

[0057]

[0058]

[0059] Based on the Pichia pastoris genome sequence, upstream and downstream fragments of the protease M1-M9 genes were obtained. Protease knockout plasmids m1-m9 were constructed using the primers shown in Table 6. Taking the construction of plasmid m1 as an example, the upstream gene sequence of protease M1 was obtained by PCR using primers M1-U-F1 and M1-U-R1. The hygromycin resistance sequence covering the M1 gene was obtained by PCR using primers M1-H-F2 / R2. The downstream gene sequence of protease M1 was obtained by PCR using primers M1-D-F3 / R3. The ori gene sequence was obtained by PCR using primers M1-O-F4 / R4. The four gene sequence fragments were ligated using homologous recombinase, then electroporated into E. coli DH5α competent cells for culture and verification, yielding plasmid m1. Plasmids m1-m9 were prepared using the same method. Plasmids m1-m9 were linearized and electroporated into recombinant Pichia pastoris B competent cells to culture strains BM1-BM9. Plasmids m1-m9 were linearized and electroporated into recombinant Pichia pastoris GB to obtain strains GM1-GM9 (as shown in Table 7).

[0060] Table 6. Plasmids used to knock out proteases M1-M9

[0061]

[0062]

[0063] Table 7. Plasmid information contained in BM1-BM9 and GM1-GM9 strains

[0064]

[0065] GM1-GM9 and BM1-BM9 strains were subjected to shake-flask fermentation, respectively. 200 μL each of the fermentation broth and the supernatant from centrifugation of GM1-GM9 strains were added to 96-well plates, and fluorescence was detected using a microplate reader. The results are as follows: Figure 2 As shown in C, the fluorescence intensity of the supernatant of strains GM3 and GM8 was significantly increased while the fluorescence intensity of the fermentation broth decreased, indicating that protease M3 and protease M8 have a significant effect on inhibiting the degradation of brassinolide and its extracellular secretion.

[0066] The content of brassinolide in the fermentation broth of strains BM1-BM9 was detected using BCA. The brassinolide yield of strain BM3 was 118.6 mg / L, and that of strain BM8 was 319.2 mg / L. The results are as follows: Figure 2 As shown in D in the diagram.

[0067] Example 3: Optimizing fermentation temperature in a 3L fermenter to increase yield

[0068] The fermentation temperature of the constructed strain was optimized, and the fermentation operation steps are as follows.

[0069] (1) Preparation of seed culture: Pick a colony of recombinant Pichia pastoris B and place it in a test tube containing 2 mL of YPD medium. Incubate at 30°C and 220 r·min -1 Incubate overnight. Inoculate the overnight culture into a 500 mL shake flask containing 100 mL of YPD medium as a seed culture, and incubate at 30°C and 220 r·min. -1 Incubate for 16-20 hours until OD (Oxygen Demand) is reached. 600 Reaching 10 or above.

[0070] (2) Sterilization preparation: Place the fermentation base salt culture medium containing 4% glycerol into a 3L fermenter, and then sterilize the fermenter (sterilization conditions are 121℃, 20min).

[0071] (3) After sterilization, install the corresponding pipes while they are still hot and tighten the corresponding bottle caps.

[0072] (4) Glycerol fermentation stage: After sterilization and cooling, the temperature was adjusted to the values ​​shown in Table 8, and the pH of the fermentation base salt medium was adjusted to 5.0 with ammonia. Under aseptic conditions, 4.35 mL / L of PTM1 microsalt solution was added to the base salt medium.

[0073] Table 8 Optimization of Fermentation Temperature

[0074]

[0075] Inoculate the seed culture from the shake flasks into the fermenter at an inoculation rate of 5-10% of the initial fermentation volume. Before the seed culture begins to grow, the dissolved oxygen percentage (DO) will be close to 100%. As the culture grows, it consumes oxygen, causing the DO value to decrease. Add oxygen to maintain the DO value above 20%. Cultivate batch cultures until glycerol is completely consumed (18 to 24 hours), which is indicated by the DO value increasing to 100% (this step indicates that all glycerol in the bottom tank has been consumed; the criterion is that when the DO value and the stirring speed reach the maximum speed, cancel the automatic control and manually control the speed at the maximum of 1000 rpm, the DO value drops to 0% and begins to rebound, quickly climbing to the peak value. When the peak value remains unchanged for about 1 minute, start adding glycerol. The initial glycerol flow rate is 10 mL / h. After the addition begins, the DO value will start to decrease until it stabilizes. If the DO value is below 20% after stabilization, stop adding glycerol. After the DO value climbs to the peak value, reduce the glycerol flow rate and start adding glycerol again. After adding glycerol and seeing the DO value decrease, stop adding glycerol and see if the DO value rebounds within a few minutes or momentarily. If this phenomenon occurs, it means that the added glycerol flow rate is just enough for the growth of yeast in the tank).

[0076] (5) Glycerol feed-by-batch stage: Start feeding 50% w / v glycerol, with 12 mL of PTM1 microsalt per liter of glycerol feed (PTM1 microsalt is added to the laminar flow hood after 50% glycerol sterilization). Set the feed rate to 18.15 mL / h / L. Continue feeding glycerol for approximately four hours or longer until a cell wet weight of 180 to 220 g / L is reached at the end of this stage.

[0077] If the dissolved oxygen percentage is below 20%, glycerol feeding should be stopped, and the oxygen rate should not be increased until the dissolved oxygen percentage reaches its peak. At this point, adjustments can be made to agitation, aeration, pressure, or oxygen supply.

[0078] (6) Methanol fermentation stage: Before starting methanol feeding, all glycerol needs to be consumed (complete consumption is indicated by a DO value reaching 100%, the criterion being: after a DO value decreases, it remains stable for a period of time and then rapidly rises to 60-70%) to fully induce the transcriptional activity of the promoter of methanol oxidase 1 (AOX1). Methanol (containing 12 mL of PTM1 trace salt per liter of methanol) is added at a flow rate of 3.6 mL / h / L-4.8 mL / h / L to allow the culture to adapt to methanol. The temperatures for the methanol fermentation stage are shown in Table 8.

[0079] In the first 2-3 hours, methanol will accumulate in the fermenter. As the culture adapts to methanol, the dissolved oxygen (DO) value will be unstable, eventually stabilizing and remaining above 20%. If the DO value cannot be maintained above 20%, stop the methanol feed, wait for the DO value to reach its peak, and then continue feeding at the current methanol feed rate. Increase stirring, aeration, pressure, or oxygen supply to maintain the dissolved oxygen percentage above 20%.

[0080] When the culture is fully adapted to methanol utilization (within 2-4 hours of the start of the methanol fermentation phase) and methanol use is limited, stable DO readings and a rapid DO peak time (generally less than 1 minute) will be observed. After the culture is fully adapted to methanol, feed methanol at a rate of 4.8 mL / h / L for at least 1 hour. Then double the feed rate to approximately 7.3 mL / h / L, feed at 7.3 mL / h / L for 2 hours, and then increase the methanol feed rate to 10.9 mL / h / L. This feed rate remains constant for the remainder of the fermentation.

[0081] The fermentation time of the methanol fermentation stage is about 98 hours (the total time of the glycerol fermentation stage, the glycerol feeding stage and the methanol fermentation stage is 146 hours).

[0082] Sampling was performed at the end of each fermentation stage, at least twice daily, with 6 mL of sample taken at each time point. One mL aliquot was then taken from each 6 mL sample. Cell growth was analyzed using OD0.05. 600 (Assessment was performed based on cell wet weight), and samples were stored on ice. Process data from the 3L fermenter are as follows: Figure 3 As shown.

[0083] Using BCA to detect the content of brassinolide in the fermentation broth, it was found that variable temperature fermentation (i.e., group C in Table 8) during the fermentation process in a 3L fermenter was more conducive to the production of brassinolide by Pichia pastoris, with a yield of 426 mg / L.

[0084] Example 4: Optimization of culture medium formulation and verification of BM8 strain yield scale-up

[0085] The formulation of the basal salt medium was optimized to further increase the yield of Brazil gluten. The optimized basal salt medium formulation is shown in Table 9.

[0086] Table 9 Optimized basal salt culture medium formulation

[0087]

[0088] The fermentation process is as follows.

[0089] (1) Preparation of seed culture: Pick recombinant Pichia pastoris B and place it in a test tube containing 2 mL of YPD medium. Incubate at 30°C and 220 r·min-1 Incubate overnight. Inoculate the overnight culture into a 500 mL shake flask containing 100 mL of BMGY medium as a seed culture, and incubate at 30°C and 220 r·min. -1 Incubate for 16-20 hours until OD (Oxygen Demand) is reached. 600 Reaching 10 or above.

[0090] (2) Sterilization preparation: Place the basic salt culture medium containing 4% glycerol into the fermenter, and then sterilize the fermenter (sterilization conditions are 121℃, 20min).

[0091] (3) After sterilization, install the corresponding pipes while they are still hot and tighten the corresponding bottle caps.

[0092] (4) Glycerol fermentation stage: After sterilization and cooling, set the fermentation temperature to 30°C, stir and aerate to the operating conditions (usually the maximum rpm and 0.1-1.0 vvm of air), and adjust the pH of the basal salt medium to 5.0 with ammonia. Aseptically add 4.35 mL / L of PTM1 trace salt to the basal salt medium.

[0093] Inoculate the seed culture from the shake flasks into the fermenter at an inoculation rate of approximately 5-10% of the initial fermentation volume. The dissolved oxygen (DO) value is close to 100% before the seed culture begins to grow. As the culture grows, it consumes oxygen, causing the DO value to decrease; maintain the DO value above 20% by adding oxygen.

[0094] Cultivate until glycerol is completely consumed (18 to 24 hours), indicated by a DO value increasing to 100%. (This step is to determine when all glycerol in the bottom tank is consumed. The criterion is that when the DO value and the stirring speed reach their maximum, cancel automatic control and manually control the speed to the maximum of 1000 rpm. When the DO value drops to 0% and begins to rebound, rapidly climbing to the peak value and remaining constant at the peak value for about 1 minute, start adding glycerol. The initial glycerol flow rate is 10 mL / h. After the addition begins, the DO value will start to decrease until it stabilizes. If the DO value is below 20% after stabilization, stop adding glycerol. After the DO value climbs to the peak value, reduce the glycerol flow rate and start adding glycerol again. After adding glycerol and seeing the DO value decrease, stop adding glycerol and observe whether the DO value rebounds within a few minutes or momentarily. If this phenomenon occurs, it means that the added glycerol flow rate is just enough for the yeast growth in the tank.)

[0095] (5) Glycerol + Methanol batch feeding stage: Start simultaneous feeding of 60% w / v glycerol + 40% w / v methanol, with 12 mL of PTM1 trace salt per liter of glycerol feed (trace salt is added in the laminar flow hood after sterilization of 50% glycerol), and the glycerol feed rate is set to 18.15 mL / h / L. The methanol flow acceleration is 2.2 + 0.016t (g / L) (t is time, calculated in hours. If the dissolved oxygen percentage cannot be maintained normally at >20% or fluctuates drastically for a period of time after adjusting the flow rate, the flow rate before adjustment should be restored).

[0096] Feed with glycerol and methanol for approximately four hours or longer. The cell wet weight should reach 180 to 220 g / L at the end of this phase.

[0097] If the dissolved oxygen percentage is below 20%, the feeding of glycerol or methanol should be stopped, and the oxygen rate should not be increased until the dissolved oxygen percentage reaches its peak. At this point, adjustments can be made to agitation, aeration, pressure, or oxygen supply.

[0098] (6) Methanol fermentation stage: Before starting methanol feeding, all glycerol needs to be consumed to fully induce the transcriptional activity of the promoter of alcohol oxidase 1 (AOX1), and methanol is added slowly. The temperature of the methanol fermentation stage is 28℃.

[0099] The glycerol feed was terminated, and induction was initiated by starting a methanol feed containing 12 mL of PTM1 trace salt (12 mL of PTM1 trace salt per liter of methanol was added under aseptic conditions). The feed rate was set to 3.6 mL / h / L.

[0100] During the initial 2-3 hours, methanol will accumulate in the fermenter, and the dissolved oxygen percentage (DO) will become unstable as the culture adapts to the methanol. Eventually, the DO reading will stabilize and remain above 20%. If the DO cannot be maintained above 20%, stop the methanol feed, wait for the DO to reach its peak, and then continue at the current methanol feed rate. Increase agitation, aeration, pressure, or oxygen supply to maintain the dissolved oxygen percentage above 20%.

[0101] Once the culture is fully adapted to methanol utilization (2–4 hours) and methanol use is limited, stable DO readings and a rapid DO peak time (generally less than 1 minute) will be observed. After adaptation, methanol is fed at a rate of 4.8 mL / h for at least 1 hour. The feed rate is then doubled to approximately 7.3 mL / h / L, and after 2 hours at a feed rate of 7.3 mL / h / L, the methanol feed rate is increased to 10.9 mL / h / L. This feed rate remains constant for the remainder of the fermentation.

[0102] Fermentation was completed after 98 hours of methanol induction. The fermentation was then stopped, and the content of brassinolide in the fermentation broth was determined using BCA. Process data from the 3L fermenter are as follows: Figure 4 As shown, the yield of Brazilian sweet protein was 726.4 mg / L.

[0103] BM8 was fermented in a 3L fermenter under the above conditions, and the final BCA yield was measured to be 4.01 g / L.

[0104] Comparative Example 1: Effect of protease combinatorial knockout on the expression of brassinolide in Pichia pastoris

[0105] The proteases in Example 2 were combined and knocked out based on the results of knockout fermentation to construct the strains shown in Table 10.

[0106] Table 10. Detailed information on combined protease knockout strains

[0107]

[0108] During the inverted plate culture process, it was found that ZM301, ZM306, ZM309, ZM801, ZM802, ZM805, and ZM809 could not grow single colonies normally. After ruling out experimental operation problems, it was proved that the double knockout of the protease combination affected the normal growth of the strain. The reason is considered to be that the knockout of multiple proteases prevented the protein degradation that Pichia pastoris should have carried out normally, affecting the normal metabolic growth of the strain and leading to the death of the strain.

[0109] The remaining strains ZM302, ZM304, ZM305, ZM307, ZM308, ZM804, ZM806, and ZM807 were subjected to shake fermentation, with the actual operation steps being the same as in Example 4. The fermentation broth was subjected to fluorescence detection and OD was measured using a UV spectrophotometer. 600 The OD of ZM302, ZM304, ZM308, and ZM804 was found. 600 The fluorescence intensity was significantly lower than that of normal strains, indicating that these knockout combinations affected the normal growth of the strains during fermentation, and there was no significant difference in fluorescence intensity compared to the single combination (e.g., Figure 5 (As shown).

[0110] ZM305, ZM307, ZM806, and ZM807 showed normal growth, with extracellular fluorescence intensity higher than that of GM5, GM6, and GM7, but not reaching the fluorescence intensity of GM3 and GM8 (e.g., Figure 5 (As shown). Therefore, the optimal protease knockout strategy is still to knock out the M8 protease alone.

[0111] Comparative Example 2: Conventional fermentation of recombinant Pichia pastoris strain in a 3L tank

[0112] The constructed strain was subjected to conventional fermentation in a 3L fermenter, and the operating steps are shown below. Finally, BCA was used to determine the yield, and the brassinolide protein yield was found to be 300 mg / L. The results are as follows. Figure 6 As shown.

[0113] The standard fermentation steps are as follows:

[0114] (1) Preparation of seed culture: Pick recombinant Pichia pastoris B and place it in a test tube containing 2 mL of YPD medium. Incubate at 30°C and 220 r·min -1 Incubate overnight. Inoculate the overnight culture solution into a 500 mL shake flask containing 100 mL of YPD medium as a seed culture, and incubate at 30°C and 220 r·min. -1 Incubate for 16-20 hours until OD (Oxygen Demand) is reached. 600 Reaching 10 or above.

[0115] (2) Sterilization preparation: Place the fermentation base salt culture medium containing 4% glycerol into the fermenter, and then sterilize the fermenter (sterilization conditions are 121℃, 20min).

[0116] (3) After sterilization, install the corresponding pipes while they are still hot and tighten the corresponding bottle caps.

[0117] (4) Glycerol fermentation stage: After sterilization and cooling, set the fermentation temperature to 30°C, stir and aerate to the operating conditions (usually maximum rpm and 0.1-1.0 vvm air), and adjust the pH of the fermentation basal salt medium to 5.0 with ammonia. Aseptically add 4.35 mL / L PTM1 trace salt to the basal salt medium.

[0118] Inoculate the seed culture from the shake flasks into the fermenter at an inoculum of approximately 5-10% of the initial fermentation volume. The dissolved oxygen (DO) will be close to 100% before the seed culture begins to grow. As the culture grows, it consumes oxygen, causing the DO to decrease. Maintain the DO above 20% by adding oxygen.

[0119] Cultivate batch cultures until glycerol is completely consumed (18 to 24 hours), which is indicated by DO increasing to 100% (this step indicates that all glycerol in the bottom tank has been consumed; the criterion is that when DO + stirring speed reaches the maximum speed, cancel automatic control and manually control the speed at the maximum of 1000 rpm, DO drops to 0% and begins to rebound, quickly climbing to the peak value, and when the peak value remains unchanged for about 1 minute, start adding glycerol. The initial glycerol flow rate is 10 mL / h. After the addition begins, DO will start to decrease until it stabilizes. If DO stabilizes and is below 20%, stop adding glycerol. After DO climbs to the peak value, reduce the glycerol flow rate and start adding glycerol again. After adding glycerol and seeing DO decrease, stop adding glycerol and see if DO rebounds within a few minutes or momentarily. If this phenomenon occurs, it means that the added glycerol flow rate is just enough for the growth of yeast in the tank).

[0120] (5) Glycerin batch feeding stage: Start feeding 50% w / v glycerin, with 12 mL of PTM1 trace salt per liter of glycerin feed (trace salt is added to the laminar flow hood after 50% glycerin sterilization) and set the feed rate to 18.15 mL / h / L.

[0121] Glycerin feeding should continue for approximately four hours or longer. At the end of this phase, a cell yield of 180 to 220 g / L wet cells should be achieved. If the dissolved oxygen percentage falls below 20%, glycerin feeding should be stopped, and the oxygen rate should not be increased until the dissolved oxygen percentage reaches its peak. At this point, adjustments can be made to agitation, aeration, pressure, or oxygen supply.

[0122] (6) Methanol fermentation stage: Before starting methanol feeding, all glycerol needs to be consumed to fully induce the transcriptional activity of promoter AOX1.

[0123] The glycerol feed was terminated, and induction was initiated by starting a methanol feed containing 12 mL of PTM1 trace salt (12 mL of trace salt was added to each liter of methanol under aseptic conditions). The methanol feed rate was set to 3.6 mL / h / L.

[0124] During the initial 2-3 hours, methanol will accumulate in the fermenter, and the dissolved oxygen percentage (DO) will become unstable as the culture adapts to the methanol. Eventually, the DO reading will stabilize and remain above 20%. If the DO cannot be maintained above 20%, stop the methanol feed, wait for the DO to reach its peak, and then continue at the current methanol feed rate. Increase agitation, aeration, pressure, or oxygen supply to maintain the dissolved oxygen percentage above 20%.

[0125] Once the culture is fully adapted to methanol utilization (2–4 hours) and methanol use is limited, stable DO readings and a rapid DO peak time (typically less than 1 minute) will be observed. After adaptation, methanol is fed at a rate of 4.8 mL / h for at least 1 hour, then the feed rate is doubled to approximately 7.3 mL / h / L. After 2 hours of feeding at 7.3 mL / h / L, the methanol feed rate is increased to 10.9 mL / h / L. This feed rate is maintained for the remainder of the fermentation.

[0126] Fermentation was continued for 98 hours until methanol induction, at which point the fermentation was terminated. The content of brassinolide in the fermentation broth was detected by BCA, and the yield of brassinolide was 300 mg / L.

[0127] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant Pichia pastoris, characterized in that, The Pichia pastoris heterologous expression gene sequence is shown in SEQ ID NO.1 as Brazil sweet protein, and the amino acid sequence of the protease shown in SEQ ID NO.3 is knocked out.

2. The recombinant Pichia pastoris according to claim 1, characterized in that, The expression of the Brazilian sweet protein was initiated using the AOX1 promoter.

3. The recombinant Pichia pastoris according to claim 1, characterized in that, The Pichia pastoris strain is Pichia pastoris X33.

4. A microbial agent comprising the recombinant Pichia pastoris according to any one of claims 1-3.

5. The use of the recombinant Pichia pastoris according to any one of claims 1-3 or the microbial agent according to claim 4 in the production of Brazil gluten.

6. A method for producing Brazil protein, characterized in that, The recombinant Pichia pastoris according to any one of claims 1-3 or the microbial agent according to claim 4 is inoculated into a fermentation medium for fermentation and culture, and the expression of Brazil protein is induced by methanol.

7. The method according to claim 6, characterized in that, The fermentation medium includes CaSO4, K2SO4, MgSO4·7H2O, KOH, glycerol and H3PO4, wherein each liter of fermentation medium contains 10-20 mL of H3PO4.

8. The method according to claim 6, characterized in that, During the first 48 hours of fermentation, the fermentation temperature was 30-31℃, and during the 48th-146th hours of fermentation, the fermentation temperature was 27-28℃.

9. The method according to claim 6, characterized in that, During the 24th to 48th hour of fermentation, fed-batch fermentation was carried out using glycerol and methanol, with a volume ratio of glycerol to methanol of (6-7):(3-4).

10. The method according to claim 9, characterized in that, When the bacterial wet weight reaches 180g / L-220g / L, stop the fed-feed fermentation. After stopping the feeding of glycerol and methanol for 1-2 hours, add methanol to induce fermentation.

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