Saccharomyces cerevisiae engineering bacterium for efficiently expressing dye decolorizing peroxidase and application thereof

Abstract

The invention discloses saccharomyces cerevisiae engineering bacteria capable of efficiently expressing dye decolorizing peroxidase and application of the saccharomyces cerevisiae engineering bacteria. The brevibacillus brevis BaDyP is heterologously expressed in saccharomyces cerevisiae, a strain BLWJ001 capable of synthesizing the BaDyP is constructed, the strain BLWJ001 is taken as an original strain, Hem14, Hem15, Hem2, Hem13, Hem1, PUG1, SPS22, REE1, PHO84, CLB2 and Hem4 are knocked in or delta shm1, delta gcv1 and delta HMX1 are knocked out, the heme yield is increased, the decolorization rate of the engineering strain for dyes is greatly improved, and a solid foundation is laid for research and application of decolorization in the fields of food, environment, chemical engineering and the like. In addition, rational design and site-directed mutagenesis modification are carried out on BaDyP, so that the catalytic performance and stability of BaDyP are improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and environment, and relates to an engineering yeast of saccharomyces cerevisiae capable of efficiently expressing dye decolorizing peroxidase and an application thereof. Background Art

[0002] Dye-decolorizing peroxidases (EC 1.11.1.19, abbreviated as DyPs) are a class of heme-containing microbial peroxidases. DyPs were first identified in 1999 by Kim et al. from Geotrichum candidum Dec1. Unlike traditional peroxidases, DyPs not only catalyze the reduction of hydrogen peroxide to water but also efficiently oxidize a variety of dye substrates, thus possessing broad application potential in fields such as papermaking, coatings, environmental remediation, and bioenergy. In recent years, researchers have focused on the degradation capabilities of microbial DyPs, particularly their unique advantages in the degradation of dyes and lignin. Although the structure and enzymatic properties of DyPs have been extensively studied, the specific mechanisms by which they catalyze the degradation of dyes and lignin remain largely undefined. Nevertheless, their value in industrial and environmental applications has been widely recognized.

[0003] Currently, DyPs face numerous challenges in industrial applications, including low enzyme activity, exogenous enzyme contamination, complex purification processes, long production cycles, and high costs. In recent years, researchers have focused on optimizing the catalytic efficiency of DyPs and have made significant progress in metabolic engineering and elucidating catalytic mechanisms. Heme, a key cofactor of DyPs, influences their catalytic activity. However, low intracellular free heme levels limit the efficient expression of heme-dependent enzymes. Therefore, increasing heme synthesis is a key strategy to enhance the industrial application potential of DyPs.

[0004] Currently, anthraquinone dyes have disadvantages such as difficulty in degradation and high toxicity, which makes the treatment of printing and dyeing wastewater face many difficulties. The present invention aims to provide a BaDyP enzyme heterologously expressed in Saccharomyces cerevisiae and metabolically engineer it to realize its application in the fields of food, environment, chemical industry, etc. Summary of the Invention

[0005] The present invention aims to provide an engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses a dye-decolorizing peroxidase and its application. The present invention relates to heterologous expression, metabolic engineering, enzyme engineering modification and application of the dye-decolorizing peroxidase BaDyP.

[0006] The technical ideas of the present invention are as follows: (1) The present invention is based on the dye decolorization peroxidase (BaDyP) derived from Brevibacillus agri DSM6348, and verifies its efficient oxidation ability in dye decolorization. Through bioinformatics analysis and experimental verification, it was found that BaDyP has strong oxidative activity and wide substrate adaptability, belongs to a unique heme peroxidase family, and has high application potential in the degradation of complex pollutants. In addition, Brevibacillus itself has strong stress resistance and can grow stably in adverse environments, thereby improving the feasibility of BaDyP in actual industrial applications. (2) Using a metabolic engineering strategy, BaDyP is heterologously expressed in brewer's yeast (Saccharomyces cerevisiae BJ5464-NpgA), and by optimizing the heme synthesis pathway, the biosynthesis level of heme during the BaDyP expression process is increased, thereby significantly enhancing its decolorization efficiency for dyes. In addition, rational design and site-directed mutagenesis are carried out on BaDyP to improve its catalytic performance and stability.

[0007] The technical solution adopted in the present invention is as follows:

[0008] 1. BaDyP of Bacillus brevis was heterologously expressed in Saccharomyces cerevisiae to construct the strain BLWJ001 that can synthesize BaDyP (heme production of 0.86 mg / L). Then, using strain BLWJ001 as the starting strain, Hem14 and Hem15 were knocked in separately to construct the engineered Saccharomyces cerevisiae strain LWJ001, with a heme production of 2.53 mg / L.

[0009] 2. Using strain BLWJ001 as the starting strain, Hem2 and Hem13 were knocked in individually to construct the Saccharomyces cerevisiae engineered strain LWJ002, with a hemoglobin production of 2.15 mg / L.

[0010] 3. Using strain BLWJ001 as the starting strain, single knockout of Δshm1 was constructed to construct the engineered strain LWJ003, with a hemoglobin production of 1.13 mg / L.

[0011] 4. Using strain BLWJ001 as the starting strain, Hem14, Hem15, Hem2 and Hem13 were knocked in to construct the Saccharomyces cerevisiae engineered strain LWJ004, with a hemoglobin production of 3.87 mg / L.

[0012] 5. Using strain BLWJ001 as the starting strain, Hem14, Hem15, Hem2, Hem13 were knocked in and Δshm1 was knocked out to construct the Saccharomyces cerevisiae engineered strain LWJ005, with a hemoglobin production of 3.94 mg / L.

[0013] 6. Based on the cerevisiae engineered strain LWJ005, Hem1 was overexpressed to construct the cerevisiae engineered strain LWJ006, with a hemoglobin production of 12.23 mg / L.

[0014] 7. Based on the brewer's yeast engineered strain LWJ006, Δgcv1 was added to construct the brewer's yeast engineered strain LWJ007, with a hemoglobin production of 21.16 mg / L.

[0015] 8. Based on the engineered Saccharomyces cerevisiae strain LWJ007, ΔHMX1 was added to construct the engineered Saccharomyces cerevisiae strain LWJ008, with a hemoglobin production of 25.27 mg / L.

[0016] 9. Based on the Saccharomyces cerevisiae engineered strain LWJ008, PUG1 was overexpressed to construct the Saccharomyces cerevisiae engineered strain LWJ009, with a hemoglobin production of 26.75 mg / L.

[0017] 10. Based on the Saccharomyces cerevisiae engineered strain LWJ009, SPS22 and REE1 were overexpressed to construct the Saccharomyces cerevisiae engineered strain LWJ010, with a hemoglobin production of 30.03 mg / L.

[0018] 11. Based on the cerevisiae engineered strain LWJ010, PHO84 and CLB2 were overexpressed to construct the cerevisiae engineered strain LWJ011, with a hemoglobin production of 32.66 mg / L.

[0019] 12. Based on the cerevisiae engineered strain LWJ011, Hem4 was overexpressed to construct the cerevisiae engineered strain LWJ012, with a hemoglobin production of 33.21 mg / L.

[0020] 13. Perform any of the following point mutations on the heterologously expressed Bacillus brevis BaDyP in the engineered yeast LWJ012: mutation site A253I (corresponding to the complete base sequence of SEQ ID NO. 2), mutating position 253 from alanine (Ala, GCA) to isoleucine (Ile, ATA);

[0021] The mutation site is P307W (the corresponding complete base sequence is SEQ ID NO. 3), which mutates position 307 from proline (Pro, CCA) to tryptophan (Trp, TGG);

[0022] The mutation site is H335F (the corresponding complete base sequence is SEQ ID NO. 4), and position 335 is mutated from histidine (His, CAT) to phenylalanine (Phe, TTT).

[0023] The present invention employed the above technical solution to heterologously express Bacillus brevis BaDyP (SEQ ID NO. 1, codon-optimized and gene-synthesized sequence of Saccharomyces cerevisiae) within S. cerevisiae BJ5464-NpgA through genetic engineering, successfully constructing the strain BLWJ001 (WT) capable of synthesizing BaDyP. The nucleotide sequence of the BaDyP gene is shown in SEQ ID NO. 1. The obtained BLWJ001 strain was further subjected to metabolic engineering transformation, and Hem14 (NCBI Gene ID: 856733), Hem15 (NCBI Gene ID: 854347), Hem2 (NCBI Gene ID: 852842), Hem13 (NCBI Gene ID: 851614), Hem1 (NCBI Gene ID: 851818), PUG1 (NCBI Gene ID: 856934), SPS22 (NCBI Gene ID: 850309), REE1 (NCBI Gene ID: 853238), PHO84 (NCBI Gene ID: 854916), CLB2 (NCBI Gene ID: 856236), and Hem4 (NCBI Gene ID: 854452) were knocked in, and Δshm1 (NCBI Gene ID: 852565), Δgcv1 (NCBI Gene ID: 851582), and ΔHMX1 (NCBI Gene ID: 851614) were knocked in. ID: 850902). The heme production of the metabolically engineered strain was measured. Finally, the metabolically engineered strain was cultured and crushed to obtain a crude enzyme solution. The crude enzyme solution was used to perform a decolorization reaction with industrial printing and dyeing wastewater. Meanwhile, a point mutation was performed on BaDyP. Due to the use of the above technical solution, the present invention has the following beneficial effects:

[0024] 1. This invention enhances heme biosynthesis and the catalytic efficiency of BaDyP in S. cerevisiae BJ5464-NpgA through genetic and protein engineering. First, through metabolic engineering, Hem14, Hem15, Hem2, Hem13, Hem1, PUG1, SPS22, REE1, PHO84, CLB2, and Hem4 are knocked in, while Δshm1, Δgcv1, and ΔHMX1 are knocked out. This increases heme production, significantly improving the decolorization efficiency of the engineered strain against dyes. This approach lays a solid foundation for research and application in decolorization applications in the food, environmental, and chemical industries.

[0025] 2. The present invention increases the heme production of BaDyP heterologously expressed in Saccharomyces cerevisiae, and simultaneously increases the decolorization rate of dyes by BaDyP heterologously expressed in yeast engineering bacteria.

[0026] 3. The present invention also performs point mutation modification on BaDyP, a heterologously expressed Bacillus brevis in the engineered yeast LWJ012, to enhance its catalytic performance and stability. The results show that unoptimized BaDyP has limited decolorization effects in Saccharomyces cerevisiae. However, after metabolic engineering, BaDyP's decolorization ability is significantly improved due to increased heme synthesis. It is noteworthy that expressing BaDyP in Saccharomyces cerevisiae not only helps enhance its application value in the decolorization of industrial printing and dyeing wastewater, but also provides new possibilities for its decolorization applications in fields such as food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results show that after metabolic engineering, BaDyP was heterologously expressed in wild-type and engineered strains of Saccharomyces cerevisiae to increase the heme content.

[0028] Figure 2 The decolorization rates of the crude enzyme solution of BLWJ001 (WT) strain and the engineered strain on Reactive Black 5 (A), Dadan Yellow (B) and industrial printing and dyeing wastewater (C).

[0029] Figure 3 Comparison of the decolorization effects of crude enzyme solution of BLWJ001 (WT) strain and engineered strain on Reactive Black 5 (A), Dadan Yellow (B) and industrial printing and dyeing wastewater (C).

[0030] Figure 4 The decolorization rate of Reactive Black 5, Dadan Yellow and industrial printing and dyeing wastewater was studied by the crude enzyme solution of point mutation engineering strain LWJ012. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. That is, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are commercially available unless otherwise specified; the equipment used are all conventional experimental equipment unless otherwise specified.

[0033] 1. Experimental Methods

[0034] 1. Preparation of DNA Fragments and Plasmid Construction

[0035] In the present invention, the BaDyP target gene was constructed on the pXW55 vector from the perspective of genetic engineering, that is, the pXW55-BaDyP plasmid was constructed. A primer pair containing homology arms was designed to amplify the target gene, and the target gene was connected to the promoter and terminator by PCR. The target fragment was recovered and purified by DNA gel. The purified fragment was ligated by Gibson recombination and transformed into Escherichia coli. The Escherichia coli transformation steps are as follows: (1) Take out the Escherichia coli heat shock competent cells and place them on ice for 5 minutes; (2) Add 5μl of ligation product and ice bath for 30 minutes; (3) Heat shock in a 42℃ metal bath for 90 seconds and immediately take out and ice bath for 5 minutes; (4) Add 600μl of LB culture medium and culture at 37℃, 150rpm for 60 minutes; (5) Spread the culture solution on ampicillin-resistant LB solid culture medium and culture it in a 37℃ incubator for 16 to 24 hours; Pick out the positive clones to extract the plasmid and send it for sequencing verification.

[0036] 2. Gene knock-in and knock-out using the CRISPR-Cas9 system

[0037] 2.1 Gene knock-in method

[0038] (1) Design specific primers to amplify 500 bp homology arm sequences upstream and downstream of the target gene;

[0039] (2) The expression cassette (TDH3p-XX-CYC1t or TEF1p-XX-ADH1t) was connected to the vector using multi-fragment cloning technology and transformed into E. coli DH5α competent cells;

[0040] (3) The positive clones were verified by sequencing and the recombinant gene fragment (Donor) was obtained by enzyme digestion.

[0041] 2.2 Gene knockout method

[0042] (1) Connect the upstream and downstream homology arms by overlap extension PCR;

[0043] (2) The Donor fragment was recovered and purified by agarose gel electrophoresis.

[0044] 2.3sgRNA design and plasmid construction

[0045] (1) Use the CHOPCHOP online tool (http: / / chopchop.cbu.uib.no / ) to design sgRNA targeting the N20 sequence;

[0046] (2) The N20 sequence was integrated into the sgRNA vector by designing primers, PCR and DNA recombination ligation technology.

[0047] 2.4 Construction of recombinant strains

[0048] The Donor fragment, sgRNA expression plasmid, and Cas9 expression plasmid were co-transformed into the host bacteria to obtain the following engineered strains:

[0049] LWJ001: Single knock-in of TDH3p-Hem14-CYC1t-TEF1p-Hem15-ADH1t at the 416d site.

[0050] LWJ002: Single knock-in of TDH3p-Hem2-CYC1t-TEF1p-Hem13-ADH1t at the 1021a site.

[0051] LWJ003: Δshm1 single knockout.

[0052] LWJ004: Knock-in Hem14, Hem15, Hem2, and Hem13 simultaneously.

[0053] LWJ005: LWJ004 with Δshm1 knocked out.

[0054] LWJ006: Based on LWJ005, TDH3p-Hem1-CYC1t was knocked into the X1 site.

[0055] LWJ007: LWJ006 with Δgcv1 knocked out.

[0056] LWJ008: LWJ007 with ΔHMX1 knocked out.

[0057] LWJ009: Based on LWJ008, TEF1p-PUG1-ADH1t was knocked into the X2 site.

[0058] LWJ010: Based on LWJ009, TDH3p-SPS22-CYC1t-TEF1p-REE1-ADH1t was knocked in at the X3 locus. LWJ011: Based on LWJ010, TDH3p-PHO84-CYC1t-TEF1p-CLB2-ADH1t was knocked in at the XI-1 locus.

[0059] LWJ012: Based on LWJ011, TDH3p-Hem4-CYC1t was knocked into the XI-2 site.

[0060] Note: The correctness of gene editing in all strains was verified by PCR and sequencing.

[0061] 3. Verification of gene knock-in and knock-out strains

[0062] (1) Transform the pCas9 plasmid into Saccharomyces cerevisiae to produce competent cells containing pCas9;

[0063] (2) The Donor fragment and gRNA were simultaneously transformed into competent cells, spread on (G418+NAT) solid plate medium (G418+NAT), and cultured at 30°C for 3 to 4 days;

[0064] (3) Single clones were picked and colony PCR was performed to verify whether the gene was knocked in or knocked out;

[0065] (4) Transfer a single colony with the target gene knocked in or knocked out to 5 mL of YPD liquid medium, culture overnight, and then streak onto YPD medium and culture at 30°C and 220 rpm for 48 h.

[0066] (5) Copy the streaked culture medium onto YPD solid plate medium (NAT) and YPD solid plate medium (G418), and select the strains that do not grow on YPD solid plate medium (NAT) but grow on YPD solid plate medium (G418) to prepare them into competent state.

[0067] 4. Preparation of E. coli DH5α competent cells

[0068] (1) Streak the strain stored at -80℃ onto an LB solid plate and culture it in a 37℃ constant temperature incubator overnight.

[0069] (2) Pick a single colony and inoculate it into 2 mL of LB liquid medium. Incubate overnight at 37°C in a shaker at 220 rpm.

[0070] (3) Pipette 0.5 mL of bacterial solution and transfer it to 50 mL / 250 mL LB liquid medium. Cultivate the bacterial solution at 37°C in a shaker at 220 rpm. 600 To 0.4-0.6.

[0071] (4) Centrifuge the bacterial solution in a 50 mL sterile centrifuge tube at 4°C, 3500 rpm for 10 min. Discard the supernatant and add 35 mL of ice-cold 0.1 mol / L CaCl2 to resuspend the cells. After an ice bath for 10 min, centrifuge at 4°C, 3500 rpm for 10 min. Discard the supernatant and repeat this step once.

[0072] (5) Add 2 mL of ice-cold 0.1 mol / L CaCl2 (containing 20% glycerol) to resuspend the cells and incubate on ice for 10 min. Pipette 100 μL of the solution into 1.5 mL centrifuge tubes and store at -80°C until needed.

[0073] 5. Preparation of Competent Saccharomyces cerevisiae

[0074] Refer to the instructions of the Frozen-EZ Yeast Transformation II Kit from Zymo Research to prepare competent Saccharomyces cerevisiae cells. The specific steps are as follows:

[0075] (1) The yeast strain stored at -80°C was streaked onto YPD solid plate medium and cultured at 28°C for 2 days.

[0076] (2) Pick a single colony with good growth on the plate and inoculate it into 5 mL of YPD liquid medium. Incubate at 28°C and 220 rpm for 1 day.

[0077] (3) Take 2 mL of cultured bacterial solution and transfer it to 25 mL / 250 mL YPD liquid medium at 50 °C and culture at 220 r / min until OD 600 It is 0.8-1.0.

[0078] (4) Centrifuge the bacterial solution in a sterile centrifuge tube at 500 × g for 4 minutes at room temperature and discard the supernatant.

[0079] (5) Add a certain volume of Frozen-EZ solution 1 to resuspend the cells, centrifuge at 500 × g for 4 min at room temperature, and discard the supernatant.

[0080] (6) Add a certain volume of Frozen-EZ solution 2 to resuspend the cells, aspirate 50 μL into 1.5 mL sterile centrifuge tubes, and store in a -80°C refrigerator.

[0081] 6. Recombinant Protein Expression in Saccharomyces cerevisiae

[0082] The constructed pXW55-BaDyP plasmid was used to transform yeast. The specific transformation steps are as follows: (1) Add 1.5 μl of plasmid to the competent culture of Saccharomyces cerevisiae, and then add 350 μl of EZ solution 3; (2) Place in a 30℃ incubator and incubate for 90 minutes, vortexing every 20 minutes; (3) Finally, spread the culture medium on SD-URA medium. Then, pick a positive single colony of Saccharomyces cerevisiae in 5 mL of SD-Ura liquid medium and culture it at 30℃ and 220 r / min for 1 day. Then, take 2.5 mL of seed liquid and transfer it to YPD liquid medium in a 50 mL / 250 mL triangular shake flask and culture it at 30℃ and 220 r / min for 3 days.

[0083] 7. Preparation of mutants A253I, IP307W, and H335F

[0084] Using plasmid pXW55-BaDyP as a template, site-directed mutagenesis was performed using one-step PCR. The specific mutation sites and primers were designed as follows: alanine (Ala, GCA) at position 253 of BaDyP was mutated to isoleucine (Ile, ATA); proline (Pro, CCA) at position 307 of BaDyP was mutated to tryptophan (Trp, TGG); and histidine (His, CAT) at position 335 of BaDyP was mutated to phenylalanine (Phe, TTT).

[0085] Table 1 shows the primer sequences for site-directed mutagenesis (underlined mutation sites):

[0086]

[0087] The total volume of the reaction system was 50 μL, and the specific composition is shown in Table 2.

[0088] Table 2 PCR reaction system

[0089]

[0090] Reaction conditions: 98°C pre-denaturation for 1 minute; 35 cycles of denaturation at 98°C for 30 seconds, annealing at 62°C for 20 seconds, and extension at 72°C for 3.5 minutes; extension at 72°C for 5 minutes; and storage at 4°C. After PCR, 2 μL of product was analyzed by agarose gel electrophoresis. The remaining product was purified using a gel extraction kit to purify the target gene fragment. The purified fragment was ligated by Gibson recombination. Positive clones were selected and plasmids were extracted. The mutation site was verified by sequencing.

[0091] 8. Hemoglobin concentration detection

[0092] (1) Dissolve hemin in Triton X-100 / NaOH solution (containing 25 g / L Triton X-100 and 0.1 mol / L NaOH) and obtain a hemin standard curve using HPLC.

[0093] (2) Add Triton X-100 / NaOH solution (containing 25 g / L Triton X-100 and 0.1 mol / L NaOH) to the bacteria to suspend them, then ultrasonicate them for 10 min, and determine the hemoglobin content by HPLC.

[0094] (3) The fermentation supernatant was mixed with a 10x Titon X-100 / NaOH solution (containing 250 μg Titon X-100 and 1 mol / L NaOH) at a volume ratio of 9:1 and allowed to stand for 10 min. The mixture was then mixed with anhydrous methanol at a volume ratio of 1:1 and the heme content was determined by HPLC. The total heme content was the sum of the heme content in the cell and supernatant.

[0095] 9. Preparation of Crude Enzyme Solution

[0096] (1) The fermented cells were collected by centrifugation at 8000 rpm for 10 min at 4°C and washed once with TBS pH 8.0 buffer.

[0097] (2) Resuspend the cells in TBS pH 8.0 buffer (10% of the fermentation volume) and perform ultrasonic disruption on an ice bath at a power of 450 W, operating for 2 seconds and resting for 4 seconds, for a total of 15 minutes (for S. cerevisiae, the power is 600 W, operating for 2 seconds and resting for 4 seconds, for a total of 45 minutes).

[0098] (3) Centrifuge at 4°C, 12,000 rpm for 30 min, collect the supernatant, and filter through a 0.22 μm filter membrane to obtain the crude enzyme solution.

[0099] 10. Decolorization reaction

[0100] The crude enzyme solution was subjected to a decolorization reaction with the dye. Three dyes (Reactive Black 5, Dadan Yellow, and industrial printing and dyeing wastewater) were prepared into 1 mM dye solutions using TBS buffer. The absorbance was measured at the wavelength corresponding to the maximum absorption peak of each dye, with deionized water as the control. The decolorization rate was calculated as follows: Decolorization rate (%) = (C0-C t ) / C0×100%, where C0 and C t The values represent the absorbance at the initial reaction time and at the end of the reaction, respectively. A control group used BaDyP enzyme inactivated by high temperature. The decolorization rates for the experimental group are calculated by subtracting the decolorization rate for the control group. Experiments for both the control and experimental groups were repeated three times. The reaction volume consisted of 700 μl of crude enzyme solution and 300 μl of dye in a 1 ml volume. The reaction was conducted at 30°C and 300 rpm for 12 h. This method was also used for wastewater decolorization.

[0101] 2. Experimental Results

[0102] By genetic engineering, Bacillus brevis BaDyP (SEQ ID NO. 1) was heterologously expressed in S. cerevisiae BJ5464-NpgA, and the strain BLWJ001 (WT) capable of synthesizing BaDyP was successfully constructed. The obtained BLWJ001 strain was further subjected to metabolic engineering transformation, and Hem14 (NCBI Gene ID: 856733), Hem15 (NCBI Gene ID: 854347), Hem2 (NCBI Gene ID: 852842), Hem13 (NCBI Gene ID: 851614), Hem1 (NCBI Gene ID: 851818), PUG1 (NCBI Gene ID: 856934), SPS22 (NCBI Gene ID: 850309), REE1 (NCBI Gene ID: 853238), PHO84 (NCBI Gene ID: 854916), CLB2 (NCBI Gene ID: 856236), and Hem4 (NCBI Gene ID: 854452) were knocked in, and Δshm1 (NCBI Gene ID: 852565), Δgcv1 (NCBI Gene ID: 851582), and ΔHMX1 (NCBI Gene ID: 851614) were knocked in. ID: 850902). Heme production was measured in the metabolically engineered strains. Finally, the successfully engineered strains were cultured and then crushed to obtain a crude enzyme solution. This crude enzyme solution was then used to decolorize industrial printing and dyeing wastewater. Simultaneously, a point mutation in BaDyP was introduced, significantly enhancing the decolorization effect.

[0103] The specific results are as follows:

[0104] 1. The HPLC method was used to detect the intracellular hemoglobin content of Saccharomyces cerevisiae. The hemoglobin content of the engineered bacteria was significantly increased ( Figure 1 ); the heme production in WT was 0.86 mg / L;

[0105] After single knock-in of Hem14 and Hem15, the heme production of engineered bacteria LWJ001 was 2.53 mg / L;

[0106] After single knock-in of Hem2 and Hem13, the heme production of engineered bacteria LWJ002 was 2.15 mg / L;

[0107] After single knockout of Δshm1, the heme production of engineered bacteria LWJ003 was 1.13 mg / L;

[0108] After knocking in Hem14, Hem15, Hem2, and Hem13, the heme production of engineered bacteria LWJ004 was 3.87 mg / L;

[0109] After knocking in Hem14, Hem15, Hem2, and Hem13 and knocking out Δshm1, the heme production of engineered bacteria LWJ005 was 3.94 mg / L;

[0110] The heme production of LWJ006 overexpressing Hem1 based on LWJ005 was 12.23 mg / L;

[0111] The heme production of Δgcv1 (LWJ007) was 21.16 mg / L based on LWJ006;

[0112] The heme production of ΔHMX1 (LWJ008) was 25.27 mg / L based on LWJ007;

[0113] The heme production of LWJ008-based overexpression of PUG1 (LWJ009) was 26.75 mg / L;

[0114] The heme production of LWJ009-based overexpression of SPS22 and REE1 (LWJ010) was 30.03 mg / L;

[0115] The heme production of LWJ011 overexpressing PHO84 and CLB2 was 32.66 mg / L;

[0116] The hemoglobin production of LWJ011-based overexpression of Hem4 (LWJ012) was 33.21 mg / L.

[0117] Cell biomass (OD 600 ) gradually decreased from 32.33 in WT to 19 in engineered bacteria LWJ012.

[0118] 2. The present invention improves the heme production of BaDyP heterologously expressed in Saccharomyces cerevisiae, and at the same time improves the decolorization rate of dyes by heterologously expressed BaDyP in yeast engineering bacteria. The crude enzyme solution treated with the engineering bacteria LWJ012 is used to decolorize Reactive Black 5, Dadan Yellow dye and industrial printing and dyeing wastewater. The decolorization rate of Reactive Black 5 is increased from the original 71.41% to 89.35%, and the decolorization rate of Dadan Yellow is increased from 70.12% to 87.22% ( Figure 2 、 3 );

[0119] 3. In order to realize the practical application of BaDyP decolorization in the fields of food, environment, chemical industry, etc., the crude enzyme solution was used to carry out decolorization reaction with industrial printing and dyeing wastewater, confirming the practical application value of the enzyme. The original wild-type strain had a decolorization rate of only 46.15% for industrial printing and dyeing wastewater, but the metabolically engineered BLWJ012 strain had a decolorization rate of 86.47% ( Figure 2 、 3 ).

[0120] 4. The present invention also aims to perform point mutations on BaDyP of Bacillus brevis heterologously expressed in the engineered yeast LWJ012. The mutation sites are A253I (SEQ ID NO. 2), P307W (SEQ ID NO. 3), and H335F (SEQ ID NO. 4). When the mutation site is H335F, the decolorization effect is obvious. The decolorization rate of Reactive Black 5 is increased from the original 89.35% to 95.97%, and that of Dadan Yellow is increased from the original 87.22% to 93.84%. The decolorization rate of industrial printing and dyeing wastewater is increased from the original 86.47% to 90.2%. Figure 4 ).

Claims

1. An engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses a dye-decolorizing peroxidase, characterized in that: The BaDyP of Bacillus brevis was heterologously expressed in Saccharomyces cerevisiae to construct the strain BLWJ001 capable of synthesizing BaDyP. Using the strain BLWJ001 as the starting strain, one or more of the following genes were knocked in or knocked out, or knocked in and knocked out; Knock-in gene: Hem14 、 Hem15 、 Hem2 、 Hem13 、 Hem1 、 PUG1 、 SPS22 、 REE1 、 PHO84 、 CLB2 、 Hem4 ; Knockout genes: Δshm1, Δgcv1, Δ HMX1 .

2. The Saccharomyces cerevisiae engineered bacteria that efficiently expresses dye decolorizing peroxidase according to claim 1, characterized in that: Any of the following: A; Using strain BLWJ001 as the starting strain, single knock-in Hem14 and Hem15 , construct the engineered yeast Saccharomyces cerevisiae LWJ001; B: Using strain BLWJ001 as the starting strain, single knock-in Hem2 and Hem13 , construct the engineered yeast Saccharomyces cerevisiae LWJ002; C: Using strain BLWJ001 as the starting strain, single knockout Δshm1 , constructing the engineered bacteria LWJ003; D: Using strain BLWJ001 as the starting strain, knock in Hem14 、 Hem15, Hem2 and Hem13 , construct the engineered Saccharomyces cerevisiae strain LWJ004; E: Using strain BLWJ001 as the starting strain, knock in Hem14 、 Hem15, Hem2 、 Hem13 and knockout Δshm1 , construct the brewer's yeast engineered strain LWJ005.

3. An engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses a dye-decolorizing peroxidase, characterized in that: Overexpression based on the saccharomyces cerevisiae engineered strain LWJ005 according to claim 2 Hem1 , construct the brewer's yeast engineered strain LWJ006.

4. An engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses a dye-decolorizing peroxidase, characterized in that: Based on the brewer's yeast engineered bacteria LWJ006 according to claim 3, gcv1, Construction of the engineered Saccharomyces cerevisiae strain LWJ007.

5. An engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses dye-decolorizing peroxidase, characterized in that: Based on the brewer's yeast engineered bacteria LWJ007 according to claim 4, Δ HMX1, Construction of the engineered Saccharomyces cerevisiae strain LWJ008.

6. An engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses dye-decolorizing peroxidase, characterized in that: Overexpression based on the cerevisiae engineered bacteria LWJ008 according to claim 5 PUG1 , construct the brewer's yeast engineered strain LWJ009.

7. An engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses dye-decolorizing peroxidase, characterized in that: Overexpression based on the brewer's yeast LWJ009 engineered strain according to claim 6 SPS22 、 REE1 , construct the brewer's yeast engineered strain LWJ010.

8. An engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses dye-decolorizing peroxidase, characterized in that: Overexpression based on the brewer's yeast LWJ010 according to claim 7 PHO84 、 CLB2 , construct the brewer's yeast engineered strain LWJ011.

9. An engineered yeast strain of Saccharomyces cerevisiae that efficiently expresses dye-decolorizing peroxidase, characterized in that: Overexpression based on the brewer's yeast LWJ011 according to claim 8 Hem4 , construct the brewer's yeast engineered strain LWJ012.

10. A mutant heterologously expressing Bacillus brevis BaDyP in the engineered yeast Saccharomyces cerevisiae LWJ012, characterized in that: Any of the following: A: The mutation site is A253I, which changes position 253 from alanine to isoleucine. The nucleotide sequence after the mutation is shown in SEQ ID NO.2; B: The mutation site is P307W, which changes position 307 from proline to tryptophan. The nucleotide sequence after the mutation is shown in SEQ ID NO.3; C: The mutation site is H335F, where position 335 is mutated from histidine to phenylalanine. The nucleotide sequence after the mutation is shown in SEQ ID NO.4.

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