A recombinant microorganism for high-level production of brassinoprotein and its application

Abstract

This invention belongs to the fields of biotechnology and genetic engineering, and discloses a microorganism for high-level production of sweet proteins and its applications. This invention achieves high-level production of sweet proteins by optimizing the composition of dominant expression modules, the dosage of dominant gene expression, and the protein synthesis and secretion pathways. The genetic modification disclosed in this invention can significantly increase the yield and production intensity of brassin in microbial cells, and the product is easy to extract and purify. This results in a genetically engineered bacterium with strong industrial properties and high efficiency in producing brassin, which can be applied to the industrial biomanufacturing of brassin.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and genetic engineering technology, specifically relating to a recombinant microorganism for high-level production of Brazil gluten and its applications. Background Technology

[0002] Sugar plays a vital role in our daily lives, serving as our body's energy source. However, excessive sugar intake can lead to numerous adverse effects, such as obesity, diabetes, and cardiovascular diseases like hypertension. Sweet proteins are a class of naturally occurring functional proteins found in plants. Due to their high sweetness, low calorie content, safety, non-toxicity, and biodegradability into essential amino acids, they have garnered significant attention and are considered an ideal sugar substitute. Since the 1970s, eight natural sweet proteins have been discovered in tropical plants: brazzein, miraculin, thaumatin, monellin, mabinlin, pentadin, curculin, and neoculin. However, the natural sources of these proteins are primarily located in West Africa. Their specific growing environment, long growth cycle, and complex extraction processes significantly limit the development and global commercial application of sweet proteins.

[0003] Brazzein is a small sweet protein with a molecular weight of 6.5 kDa, derived from plants in West Africa. Pentadiplandra brazzeana The fruit of the plant, brassin, is 500-2000 times sweeter than the same weight of sucrose and exhibits high stability to heat and pH. Currently, various recombinant expression systems are used to express brassin, including those from *Escherichia coli*, *Lactococcus lactis*, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Kluyveromyces lactis*, and maize. However, due to the structural characteristics of brassin itself and the limitations of production levels in various host strains, its yield has not yet met industrial production requirements, necessitating the design and creation of engineered microbial strains with high brassin production performance. Summary of the Invention

[0004] The purpose of this invention is to improve the level of carbazin secretion and expression in engineered microorganisms through genetic modification, and to create microbial strains that produce carbazin efficiently.

[0005] This invention provides a recombinant microorganism for high-level fermentation production of brassinoprotein. Preferably, the microorganism has (1) a dominant module composition for brassinoprotein expression; and (2) high-dose expression of brassinoprotein. (3) Genetic modification to improve brassinoprotein production by optimizing the synthesis and exocytosis pathways; among which, (i) The Brazil sweet protein gene expression module comprises a promoter-signal peptide-Brazil sweet protein gene-terminator connected in sequence.

[0006] (ii) In the dominant expression module, the promoter is the Pichia pastoris alcohol oxidase (AOX1) promoter; the signal peptide is the first 19 amino acids of the Pichia pastoris PAS_chr3_0030 gene; and the terminator is the Pichia pastoris dihydroxyacetone synthase (DAS1) terminator. (iii) High-dose expression of the brassinoprotein, wherein the brassinoprotein expression module is stably integrated into the microbial genome with one or two or more copies; (iv) The genetic modification targets for enhancing the exocrine pathway of Brazil sweet protein include: Upregulated expression of one, two, three, or four of the following: protein disulfide isomerase PDI1, endoplasmic reticulum oxidoreductase ERO1, molecular chaperone (KAR2), and transcription factor (HAC1). In a preferred embodiment, the genetic modification specifically includes one of the following: (i) The method for constructing the engineered strain includes one of the following: 1) Site-specific integration of genomic units; 2) Random integration of retrotransposon genome repetitive regions; (ii) The genetic modification of the protein disulfide isomerase PDI1, endoplasmic reticulum oxidoreductase ERO1, molecular chaperone (KAR2) and transcription factor (HAC1) is achieved by modifying the genetic targets of their overexpression through genetic engineering methods. In a preferred embodiment, the genetic modification specifically includes one of the following: 1) PDI1 or 2) ERO1 or 3) KAR2 or 4) HAC1 or 5) PDI1+ERO1, PDI1+KAR2, PDI1+HAC1 or 6) PDI1+ERO1+KAR2, PDI1+ERO1+HAC1 or 7) PDI1+ERO1+KAR2+HAC1 Preferably, the protein disulfide isomerase PDI1, endoplasmic reticulum oxidoreductase ERO1, molecular chaperone (KAR2), and transcription factor (HAC1) are derived from yeast, preferably from Pichia pastoris (Komagataella phaffii), Saccharomyces cerevisiae, Rhodotorula buergerianum, Pichia fermentata, Kluyveromyces marxifolia, Hansenula spp., Hansenula d'Bary, Bayer zygosacchari, Wickham spp. aberrantus, or Pichia kudria.

[0007] In addition, the amino acid sequences of the protein disulfide isomerase PDI1 are GI: 254574366, GI: 238034091 or similar, the amino acid sequences of the endoplasmic reticulum oxidoreductase ERO1 are GI: 254565069, GI: 238029441 or similar, the amino acid sequences of the molecular chaperone KAR2 are GI: 254567834, GI: 238030824 or similar, and the amino acid sequences of the transcription factor HAC1 are GI: 254565857, GI: 238029835 or similar.

[0008] The analogues described herein have one or a combination of the following characteristics: (i) A mutant of the corresponding genetic target, wherein the amino acid sequence of the mutant has at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of the corresponding genetic target; (ii) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the genetically modified protein.

[0009] Specifically, the microorganisms are fungi and bacteria; Preferably, the fungus is selected from the genera *Pichia*, *Yersinia*, *Kluyveromyces*, *Saccharomyces*, *Candida*, *Aspergillus*, or *Fusarium*. Preferably, the microorganism is Pichia pastoris (… Komagataella phaffii Yeast lipolytica, Saccharomyces cerevisiae, Rhodotorula rubra, Pichia pastoris, Kluyveromyces marx, Hansenula, Hansenula de Barry, Bayer conjugate yeast, Wickham aberrant or Pichia kudria, Trichoderma reesei or Aspergillus niger.

[0010] The present invention also provides the use of the recombinant microorganisms described herein in the production of carbapenem.

[0011] The present invention also provides a method for producing brassinolide, which involves fermenting the microorganisms in a culture medium containing a substrate to produce the brassinolide; The culture medium includes a carbon source and a nitrogen source, specifically a combination of one or more of the following: (i) The carbon source is selected from fermentable carbon sources or non-fermentable carbon sources; preferably, fermentable carbon sources include, but are not limited to, glucose, fructose, galactose, xylose, and sucrose, and non-fermentable carbon sources include, but are not limited to, ethanol, acetic acid and acetates, glycerol, lactate, methanol, and formic acid; (ii) The nitrogen source is selected from organic nitrogen sources or inorganic nitrogen sources; preferably, organic nitrogen sources include, but are not limited to, peptone, yeast powder, and corn steep liquor, and inorganic nitrogen sources include, but are not limited to, ammonium sulfate, ammonium chloride, nitrates, and ammonia.

[0012] (iii) Biomass containing carbon and nitrogen sources, including but not limited to agricultural, industrial and forestry residues or waste.

[0013] This invention first optimizes the expression module of the carbapenem gene to obtain an initial strain capable of excreting carbapenem. Subsequently, the gene dosage and expression pathway are enhanced in this strain to further improve its ability to express and secrete carbapenem. The genetic modification disclosed in this invention can significantly increase the yield and production intensity of carbapenem in microbial cells, and the product is easy to extract and purify. This results in a highly efficient genetically engineered bacterium for carbapenem production with strong industrial properties, applicable to the industrial biomanufacturing of carbapenem. Attached Figure Description

[0014] Figure 1 Schematic diagram of the Brazil sweet protein expression module.

[0015] Figure 2 The level of synthesis and secretion of brassinosteroids by recombinant Pichia pastoris containing different secretion expression elements.

[0016] Figure 3 SDS-PAGE analysis of the supernatant of fermentation broth containing recombinant Pichia pastoris with different secretion expression elements after 76 hours and 96 hours of culture.

[0017] Figure 4 The level of synthesized and secreted brassinosteroids by recombinant Pichia pastoris constructed using the retropollinated random integration method.

[0018] Figure 5 The levels of brassinosteroids secreted by recombinant Pichia pastoris with different gene doses were constructed using a site-directed integration method. Each plus sign represents the integration of one copy of the brassinosteroid secretion gene into the genome of the recombinant strain.

[0019] Figure 6 The level of brassinosteroids synthesized and secreted by recombinant Pichia pastoris with optimized protein secretion pathway.

[0020] Figure 7 SDS-PAGE analysis of the supernatant of the fermentation broth after culturing recombinant Pichia pastoris with optimized protein secretion pathway for 96 hours.

[0021] Figure 8 The level of brassin at different fermentation times when recombinant strain ST1884 was cultured in a 5-L fermenter.

[0022] Figure 9 SDS-PAGE analysis of the supernatant of the fermentation broth at different fermentation times when the recombinant strain ST1884 was cultured in a 5-L fermenter. Detailed Implementation

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] The Bradford protein concentration assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the Pichia pastoris strain and genetically modified plasmid tools were created by the inventor team of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. All culture media were prepared with distilled water unless otherwise specified.

[0026] Table 1. Amino acid and nucleotide sequences of Brazilian sweet protein used in this invention.

[0027] Table 2 Genotype information of the strains used in the examples

[0028] The GI numbers of the proteins used in the examples are as follows: the amino acid sequence of protein disulfide isomerase PDI1 is GI: 254574366, the amino acid sequence of endoplasmic reticulum oxidoreductase ERO1 is GI: 254565069, the amino acid sequence of molecular chaperone KAR2 is GI: 254567834, and the amino acid sequence of transcription factor HAC1 is GI: 254565857.

[0029] Example 1: Optimization of expression elements in the Brazil gluten secretion expression module The recombinant microbial strain carries a Brazil glycoprotein secretion expression module, such as Figure 1 As shown, the gene sequence consists of homologous arms, promoter, signal peptide, Brazil gluten protein gene, and terminator. In the specific experimental process, fusion PCR technology is used to construct the required donor DNA fragment. The promoter is one of the most important elements regulating gene expression. AOX1 Promoter (inducible) and P GAP The promoter (constitutive) is a representative promoter in the expression of exogenous proteins in Pichia pastoris. AOX1 The promoter exhibits high expression levels, is methanol-induced with a tightly regulated pattern, and is repressed by glucose, making it suitable for expressing exogenous genes; the constitutive promoter P... GAP It has broad spectrum applicability, regardless of different carbon source conditions such as glucose, glycerol, and methanol, PGAP Gene expression under promoter control can maintain a relatively stable level. Signal peptides are key guide molecules in protein synthesis, responsible for accurately introducing newly synthesized proteins into the endoplasmic reticulum lumen for folding and transporting them extracellularly via the secretory pathway. Different types of signal peptides have different abilities to act on different proteins, requiring screening and adaptation.

[0030] Therefore, different combinations of promoters and signal peptides can significantly affect the synthesis and secretion of exogenous proteins. We will use P... AOX1 With three signal peptides from different sources: MFα, MFα V50A SP 0030 The combined amino acid sequences are as follows: MFα:MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR (SEQ ID No: 3).

[0031] MFα V50A :MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDAAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR (SEQ ID No: 4).

[0032] SP 0030 :MKFAISTLLIILQAAAVFA (SEQ ID No: 5).

[0033] Using CRISPR-Cas9 technology, the protein expression module was site-directedly integrated into the neutral PNSIV-9 site in the yeast genome via electroporation. Transformants were screened using YPD resistance plates, and colony PCR was used to verify their correctness, yielding single-copy recombinant strains (Table 2): ST436, ST439, and ST441.

[0034] The recombinant Pichia pastoris strain obtained above was inoculated onto BMGY medium via YPD plates. After the cell density reached a certain level, the cell pellet was collected by centrifugation at 2000g for 5 minutes at room temperature. The supernatant of the fermentation broth was removed, and the cells were resuspended in fresh BMGY medium. The recombinant yeast strain was cultured at 30℃ and 220 rpm, with 0.5% (v / v) methanol added every 12 hours. Samples were taken after 48, 72, 96, and 120 hours of induction, and the protein concentration in the supernatant after centrifugation was determined using the Bradford assay kit.

[0035] Experimental results show that ( Figure 2The protein concentration in the fermentation broth supernatant gradually increased with the extension of induction time, showing an overall upward trend. The protein concentration reached its highest value after 120 hours of induction culture, but the cell density decreased at this point. Based on a comprehensive analysis of protein expression levels and cell state, 96 hours of induction culture was determined to be the optimal point for sample comparison. For strains ST439 and ST441, the protein synthesis and secretion levels were higher after 96 hours of induction culture, indicating that the gene expression cassette elements of these two strains have an advantage in expressing brassin. SDS-PAGE was performed on the fermentation broth supernatants after 72 and 96 hours. Figure 3 The results showed that strains ST436, ST439, and ST441 all exhibited the target band (Brazilian protein size is 6.5 kDa) within the 5-15 kDa range, indicating that the strains successfully expressed, synthesized, and secreted brassin extracellularly. According to existing literature, the signal peptide SP... 0030 It contains only 19 amino acids and can be well separated from the target protein. Furthermore, the amino acid composition of the protein expressed by strain ST441 was identified using ion mobility tandem mass-time-of-flight spectrometry (timsTOF Pro2, Bruker Daltonics). The results showed that the protein in the 5-15 kDa range of the ST441 fermentation broth supernatant was Brazil protein, and the signal peptide was completely separated from the target protein, consistent with previously reported results. Simultaneously, we constructed a constitutive promoter P... GAP Replace P AOX1 strain ST440 (P GAP ->SP 0030 ->PbBRZ->T aox1 However, the levels of brassinolide synthesis and secretion in this strain were lower than those in ST441 at all time points after fermentation. Taking all factors into consideration, strain ST441 is the dominant strain, i.e., P... AOX1 ->SP 0030 ->PbBRZ->T das1 In this experiment, the dominant expression module was induced for 96 hours.

[0036] Example 2: Construction of multi-copy strains by randomly integrating a Brazil gluten protein expression module Retropoeian genomic repetitive regions provide abundant target sites for gene integration, enabling rapid multi-copy integration and thus enhancing the expression level of exogenous genes. When constructing multi-copy strains using the targeted retropoeian genomic repetitive region random integration (Ty integration) method, fusion PCR was employed to ligate various expression elements, forming donor DNA. An appropriate amount of donor DNA was then transformed into the starting strain ST1802 via electroporation to obtain the prototrophic multi-copy recombinant strain ST2268 (Table 2).

[0037] The recombinant Pichia pastoris strain obtained above was inoculated onto SD-U plates into SD-2×SCAA medium containing 0.5% (v / v) glycerol. After the cells reached a certain density, the cell pellet was collected by centrifugation at 2000g for 5 minutes at room temperature. The supernatant of the fermentation broth was removed, and the cells were resuspended in SD-2×SCAA medium containing 0.5% (v / v) methanol. The recombinant yeast strain was cultured at 30℃ and 220 rpm, with 0.5% (v / v) methanol added every 12 hours. After 96 hours of induced expression, the supernatant of the fermentation broth was collected by centrifugation at 2000g for 10 minutes at room temperature, and the yield of extracellular protein was determined.

[0038] Experimental results show that ( Figure 4 After 96 hours of methanol-induced expression, the yield of brassinolide in the fermentation supernatant reached 73.15 mg / L. This result indicates that this method can rapidly obtain multi-copy recombinant strains expressing high levels of brassinolide, providing an effective strategy for improving the production efficiency of Pichia pastoris expression systems.

[0039] Example 3: Site-specific integration of genomic units to optimize the dosage of brassinoprotein gene Compared to random integration, targeted integration can precisely insert exogenous genes into specific sites in the genome, avoiding damage to the host gene, increasing the positive rate, and resulting in higher stability of exogenous gene expression, making it less susceptible to changes in other regions of the genome. Furthermore, since the integration site is known, it facilitates further modification of subsequent strains. In this experiment, CRISPR-Cas9 technology was used to target the integration of protein expression modules into different gene sites in the yeast genome via electroporation (the protein expression modules were constructed using fusion PCR technology as donor DNA). YPD resistance plates were used for screening, and colony PCR was used to verify the correctness of transformants. Recombinant strains with different copy numbers (1-4) were obtained (Table 2): ST495, ST496, ST1804, ST1802, and ST1821.

[0040] The recombinant Pichia pastoris strain obtained above was inoculated onto BMGY medium via YPD plates. After the cell density reached a certain level, the cell pellet was collected by centrifugation at 2000g for 5 minutes at room temperature. The supernatant of the fermentation broth was removed, and the cells were resuspended in fresh BMGY medium. The recombinant yeast strain was cultured at 30℃ and 220 rpm, with 0.5% (v / v) methanol added every 12 hours. After 96 hours of induced expression, the supernatant of the fermentation broth was collected by centrifugation at 3000g for 10 minutes at room temperature, and the yield of extracellular protein was determined.

[0041] Experimental results show that ( Figure 5After 96 hours of methanol-induced expression, strain ST1802, containing 3 copies of the target gene, exhibited the highest single-cell expression level, reaching 13.74 mg / L / OD. 600 This indicates that moderately increasing the copy number of the target protein synthesis gene within a certain range can help improve the synthesis level of the target protein. This strategy has a significant effect on increasing the production of Brazil protein synthesized and secreted by Pichia pastoris and is an effective optimization method.

[0042] Example 4: Enhancement of the protein secretion pathway When cells need to synthesize large amounts of protein, or proteins involving complex folding processes, the folding capacity of the endoplasmic reticulum (ER) may be limited, leading to the accumulation of unfolded or misfolded proteins. This protein accumulation triggers an ER stress response (UPR). If ER stress is not effectively relieved, the cell first enters cell cycle arrest, inhibiting further synthesis of abnormal proteins; however, if protein folding abnormalities persist, the cell initiates apoptosis, ultimately leading to cell lysis. Therefore, in the synthesis of recombinant proteins, correct folding is a crucial step in ensuring their successful passage through the ER and Golgi apparatus and transport to the extracellular space.

[0043] Molecular chaperones are a class of proteins that play crucial roles in protein translation, transport, folding, and modification. Protein disulfide isomerase (PDI), endoplasmic reticulum oxidase (ERO1), and endoplasmic reticulum resident chaperone protein (KAR2) promote the correct folding of target proteins, prevent the aggregation of nascent polypeptide chains, and facilitate protein secretion. Furthermore, Hac1p, a key transcription factor, binds to endoplasmic reticulum stress response elements (UPR elements) to regulate hundreds of genes related to the secretion pathway. When cells sense endoplasmic reticulum stress, Hac1p is activated, thereby regulating the expression of related genes, helping cells cope with endoplasmic reticulum stress, reducing the burden on the endoplasmic reticulum, and promoting protein synthesis and secretion.

[0044] Brazilin molecules contain four disulfide bonds (Cys4-Cys52, Cys16-Cys37, Cys22-Cys47, and Cys26-Cys49), which form the basis of its good stability, but also place higher demands on its correct folding. In this experiment, we used the inducible promoter PrFDH1 to drive PDI1, ERO1, KAR2, and HAC1, and constructed the donor DNA using fusion PCR technology. Using CRISPR-Cas9 technology, the donor DNA was integrated into the neutral site PNSI-8 via electroporation. YPD-resistant plates were used for screening, and the correctness of the transformants was verified by colony PCR. Recombinant strains were obtained (Table 2): ST1881, ST1882, ST1883, and ST1884.

[0045] The recombinant Pichia pastoris strain obtained above was cultured and treated according to the method in Example 3.

[0046] Experimental results show that ( Figure 6 After 96 hours of methanol-induced expression, the recombinant strain ST1884 overexpressing PDI showed a significantly higher yield of brassinosteroid protein than the control strain ST1851, increasing by 39%. SDS-PAGE analysis of the fermentation broth supernatant showed... Figure 7 A distinct single band was observed in the molecular weight range of 5-15 kDa, consistent with the theoretical molecular weight of brassinoprotein (6.5 kDa). This result indicates that brassinoprotein can be efficiently synthesized and secreted in recombinant strains and successfully separated from the signal peptide.

[0047] Example 5: High-density culture production of Brazilian sweet protein in a 5-L fermenter The ST1884 strain was cultured in a 5-L fermenter using a fed-batch fermentation method to achieve high-efficiency production of Brazilian sweet protein. Single colonies were picked from YPD plates and transferred to 2.5 mL of YPD medium, incubated at 30°C and 220 rpm for approximately 16 hours. After reaching a certain cell density, the cells were transferred to 50 mL of YPD medium and cultured for another 12-14 hours. Finally, the cells were transferred to a 5-L fermenter containing 1.5 L of BSM medium at an appropriate cell density. The fermentation process employed a fed-batch fermentation method, comprising three stages: glycerol batch culture, glycerol fed-batch culture, and methanol fed-batch culture.

[0048] The initial culture medium contained 4% (w / v) glycerol. During the glycerol batch culture phase, once the glycerol was depleted, a glycerol-feeding culture was performed at a feed rate of 27.23 mL / h for 4 hours. The glycerol-feeding medium consisted of 1.2% (v / v) PTM1 and 50% (w / v) glycerol. Prior to the methanol induction phase, cells underwent a 1.5-hour starvation treatment to ensure complete depletion of the glycerol in the medium. During the methanol feeding phase, a methanol solution containing 1.2% (v / v) PTM1 was used, with a feed rate ranging from 5.4 to 16.35 mL / h.

[0049] During fermentation, the temperature was maintained at 30℃, the initial stirring speed was 250 rpm, and the aeration rate was 1.0 L / min. The dissolved oxygen (DO) in the fermentation broth was maintained at no less than 20% by adjusting the aeration rate (1.5-10 L / min) and stirring speed (250-1400 rpm). Furthermore, ammonia was added to maintain the pH at 5.5, and an appropriate amount of antifoaming agent (Antifoam 204, Sigma-Aldrich, A6426) was used to suppress foaming in the fermentation broth. 5 mL samples were taken every 12 hours for OD analysis. 600 Detection, protein concentration detection.

[0050] The entire fermentation process lasted 133 hours, with a methanol induction time of 103 hours. Ultimately, the protein content in the fermentation broth supernatant was 639.11 mg / L. Figure 8 It is noteworthy that carbazin appeared as a single band in the fermentation broth supernatant. Figure 9 The results indicate that the protein has reached a high purity in the fermentation broth, and only a simple purification process is needed to obtain high-purity Brazilin. This production system utilizes a low-cost culture medium, significantly reducing production costs and improving the feasibility of commercializing Brazilin in the food industry.

Claims

1. A recombinant microorganism for high-level production of brassinolide, characterized in that, The recombinant microorganism possesses the advantageous module composition for expressing brassinoprotein; and the genetic modification enhances the exocrine expression pathway of brassinoprotein. The Brazilian sweet protein gene expression module includes a promoter-signal peptide-Brazilian sweet protein gene-terminator connected in sequence. The genetic modification to enhance the exocrine expression pathway of Brazilian sweet protein includes upregulating one or two, three or four of the following: protein disulfide isomerase PDI1, endoplasmic reticulum oxidoreductase ERO1, molecular chaperone KAR2, and transcription factor HAC1.

2. The recombinant microorganism as described in claim 1, characterized in that, In the dominant expression module, the promoter is the promoter of Pichia pastoris alcohol oxidase AOX1; the signal peptide is the first 19 amino acids of the Pichia pastoris PAS_chr3_0030 gene; and the terminator is the terminator of Pichia pastoris dihydroxyacetone synthase DAS1.

3. The recombinant microorganism as described in claim 1, characterized in that, In the dominant expression module, the Brazil sweet protein gene is stably expressed in the microbial genome with one, two, or more copies.

4. The recombinant microorganism as described in claim 1, characterized in that, The integration method is selected from: site-specific integration at genomic unit points and random integration of retrotransposon genomic repetitive regions.

5. The recombinant microorganism according to any one of claims 1-4, characterized in that, The genetic modification of the protein disulfide isomerase PDI1, endoplasmic reticulum oxidoreductase ERO1, molecular chaperone KAR2, and transcription factor HAC1 was achieved by modifying the genetic targets for their overexpression through genetic engineering methods. In a preferred embodiment, the genetic modification specifically includes one of the following: 1) PDI1 or 2) ERO1 or 3) KAR2 or 4) HAC1 or 5) PDI1+ERO1, PDI1+KAR2, PDI1+HAC1 or 6) PDI1+ERO1+KAR2, PDI1+ERO1+HAC1 or 7) PDI1+ERO1+KAR2+HAC1.

6. The recombinant microorganism as described in claim 5, characterized in that, The protein disulfide isomerase PDI1, endoplasmic reticulum oxidoreductase ERO1, molecular chaperone KAR2, and transcription factor HAC1 are derived from yeast, preferably from Pichia pastoris (…). Komagataella phaffii ), Saccharomyces cerevisiae, Rhodotorula buergerianum, Pichia pastoris, Kluyveromyces marx, Hansenula sacchari, Hansenula d'Barry, Bayer conjugate yeast, Wickham aberrant yeast or Pichia kudria. More specifically, the amino acid sequences of the protein disulfide isomerase PDI1 are GI: 254574366, GI: 238034091 or similar, the amino acid sequences of the endoplasmic reticulum oxidoreductase ERO1 are GI: 254565069, GI: 238029441 or similar, the amino acid sequences of the molecular chaperone KAR2 are GI: 254567834, GI: 238030824 or similar, and the amino acid sequences of the transcription factor HAC1 are GI: 254565857, GI: 238029835 or similar. The analogues have one or a combination of the following characteristics: (i) A mutant of the corresponding genetic target, wherein the amino acid sequence of the mutant has at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of the corresponding genetic target; (ii) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the genetically modified protein.

7. The recombinant microorganism as described in claim 5, characterized in that, The recombinant microorganisms are fungi or bacteria; Preferably, the fungus is selected from the genera *Pichia*, *Yersinia*, *Kluyveromyces*, *Saccharomyces*, *Candida*, *Aspergillus*, or *Fusarium*. Preferably, it is Pichia pastoris ( Komagataella phaffii Yeast lipolytica, Saccharomyces cerevisiae, Rhodotorula rubra, Pichia pastoris, Kluyveromyces marx, Hansenula, Hansenula de Barry, Bayer conjugate yeast, Wickham aberrant or Pichia kudria, Trichoderma reesei or Aspergillus niger.

8. The use of the recombinant microorganism according to any one of claims 1-7 in the production of brassinolide.

9. A method for preparing Brazilian sweet protein, characterized in that, The Brazilian sweet protein is produced by fermentation of the recombinant microorganism as described in any one of claims 1-7 in a culture medium containing the substrate.

10. The method as described in claim 9, characterized in that, The culture medium includes a carbon source and a nitrogen source, specifically a combination of one or more of the following: (i) The carbon source is selected from fermentable carbon sources or non-fermentable carbon sources; preferably, fermentable carbon sources include, but are not limited to, glucose, fructose, galactose, xylose, and sucrose, and non-fermentable carbon sources include, but are not limited to, ethanol, acetic acid and acetates, glycerol, lactate, methanol, and formic acid; (ii) The nitrogen source is selected from organic nitrogen sources or inorganic nitrogen sources; preferably, organic nitrogen sources include, but are not limited to, peptone, yeast powder, and corn steep liquor, and inorganic nitrogen sources include, but are not limited to, ammonium sulfate, ammonium chloride, nitrates, and ammonia. (iii) Biomass containing carbon and nitrogen sources, including but not limited to agricultural, industrial and forestry residues or waste.

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