Saccharomyces cerevisiae for relieving heme feedback inhibition and enhancing heme supply and application

Abstract

The invention discloses saccharomyces cerevisiae for relieving heme feedback inhibition and strengthening heme supply and application, and belongs to the technical field of genetic engineering. The invention constructs a 5-aminolevulinic acid synthetase mutant capable of removing heme, and the yield of 5-aminolevulinic acid and the yield of heme of a strain H5 constructed by in-situ integration of the mutant in saccharomyces cerevisiae can be increased by 1.69 times and 2.99 times respectively compared with a control strain CEN.PK2-1C. According to the present invention, the HEM1I213K and the HEM13 are further integrated on the basis of the H5 strain in the multi-copy manner, such that the heme synthesis of the obtained heme-reinforced strain H25 is improved by 16.57 times compared with the CEN.PK2-1C, the yield of the synthesized rHb and the heme binding rate are improved by 22.9% and 39.2% compared with the CEN.PK2-1C added with 25 mg / L heme, and the heme supply-reinforced and high-activity human hemoglobin synthesis can be easily achieved.

Description

Technical Field

[0001] The invention relates to a brewer's yeast capable of relieving heme feedback inhibition and strengthening heme supply and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] As a functional protein with heme as a cofactor, human hemoglobin has the ability to store and transport oxygen, and can therefore be used in the construction of artificial oxygen carriers. At present, the synthesis of recombinant human hemoglobin (rHb) using the food-safe host Saccharomyces cerevisiae as a host has the advantages of unlimited supply and avoiding pathogen contamination. However, since heme is toxic to cells, the supply of heme in natural Saccharomyces cerevisiae cells is strictly regulated. Therefore, the synthesis of physiologically active human hemoglobin in Saccharomyces cerevisiae must strengthen the supply level of heme in the host cells.

[0003] Although exogenous addition of 5-aminolevulinic acid (ALA) or heme can enhance the expression of highly active hemoglobin, the effects of these strategies are not ideal. Moderately increasing the synthesis of heme in microbial hosts can overcome these limitations. Although high-titer heme-synthesizing strains can be obtained by strengthening multiple rate-limiting enzymes in the heme synthesis pathway, the activity of key rate-limiting enzymes can be increased in Saccharomyces cerevisiae by alleviating the steric barriers of heme synthases (Hem14p and Hem15p), thereby obtaining 18.0% mol heme / mol bovine hemoglobin. However, in order to further construct a microbial platform for enhanced heme supply for efficient synthesis of highly active rHb, some problems still need to be solved, such as the feedback inhibition of heme in Saccharomyces cerevisiae on the key synthase 5-aminolevulinic acid synthase (Hem1p). This feedback inhibition phenomenon will aggravate the unnecessary waste of cellular resources during the process of heme supply enhancement, thereby adversely affecting subsequent protein synthesis.

[0004] Therefore, in order to increase the heme supply level in Saccharomyces cerevisiae while saving cellular resources as much as possible, how to alleviate the feedback inhibition of heme on the key synthetic enzyme Hem1p is an urgent problem that needs to be solved. Summary of the invention

[0005] The present invention first provides a mutant capable of relieving the feedback inhibition of heme on 5-aminolevulinic acid synthase (Hem1p), which has one or more of the following mutations based on the starting sequence:

[0006] (a) mutating the isoleucine at position 153 to alanine or glycine;

[0007] (b) mutating tyrosine at position 183 to alanine or glycine;

[0008] (c) mutating the isoleucine at position 213 to alanine, lysine or arginine;

[0009] (d) mutating phenylalanine at position 365 to alanine;

[0010] (e) The proline at position 450 was mutated to alanine.

[0011] In one embodiment, the starting sequence is shown as SEQ ID NO.1.

[0012] In one embodiment, the Hem1p alanine mutant capable of alleviating the feedback inhibition of heme includes: Hem1p I153A 、Hem1p Y183A 、Hem1p I213A 、Hem1p F365A and Hem1p P450A .

[0013] In one embodiment, the Hem1p saturation mutant capable of alleviating the feedback inhibition of heme comprises: I153G 、Hem1p Y183G 、Hem1p I213K and Hem1p I213R .

[0014] The present invention also provides a gene encoding the mutant.

[0015] In one embodiment, the encoding mutant Hem1p I213K The gene sequence is shown in SEQ ID NO.8.

[0016] The present invention also provides a recombinant microbial cell expressing the mutant.

[0017] In one embodiment, the microorganism is Escherichia coli, including but not limited to Escherichia coli BL21 (DE3).

[0018] In one embodiment, when heterologously expressed in E. coli BL21(DE3), the leader peptide (61 amino acid residues from amino acid 2 to amino acid 62 at the N-terminus) of Hem1p is removed.

[0019] In one embodiment, the mutant is expressed using pMAL-c5X as a vector.

[0020] In one embodiment, the microorganism is Saccharomyces cerevisiae, including but not limited to Saccharomyces cerevisiae CEN.PK2-1C.

[0021] In one embodiment, the starting strain of Escherichia coli is Escherichia coli BL21 (DE3).

[0022] The present invention also provides an engineered yeast strain of Saccharomyces cerevisiae that relieves the feedback inhibition of heme on Hem1p. The engineered yeast strain replaces the gene HEM1 in situ with HEM1 in the genome. I213K .

[0023] In one embodiment, the nucleotide sequence of the gene HEM1 is shown as SEQ ID NO.7.

[0024] In one embodiment, the starting strain of Saccharomyces cerevisiae includes but is not limited to Saccharomyces cerevisiae CEN.PK2-1C.

[0025] In one embodiment, the engineered Saccharomyces cerevisiae strain also overexpresses Hem3p (Gene ID: 851322; Genbank accession number: NM_001180265.1) and Hem13p (Gene ID: 851614; Genbank accession number: NM_001180352.1).

[0026] In one embodiment, the engineered yeast Saccharomyces cerevisiae has multiple copies of the HEM13 gene integrated into the rDNA locus and / or multiple copies of the HEM1 gene integrated into the Delta locus. I213K Gene.

[0027] In one embodiment, the nucleotide sequence of the HEM13 gene is as shown in SEQ ID NO.5; I213K The nucleotide sequence of the gene is shown in SEQ ID NO.8.

[0028] In one embodiment, the engineered Saccharomyces cerevisiae has integrated 7 to 9 copies of the HEM13 gene.

[0029] In one embodiment, the engineered yeast Saccharomyces cerevisiae has 8 copies of the HEM13 gene integrated, and 6 to 9 copies of the HEM1 gene integrated at the Delta site. I213K Gene.

[0030] In one embodiment, the engineered yeast Saccharomyces cerevisiae integrates 8 copies of the HEM13 gene and 9 copies of the HEM1 I213K Gene.

[0031] In one embodiment, the Saccharomyces cerevisiae uses pESC as an expression vector to express the human hemoglobin gene.

[0032] In one embodiment, the human hemoglobin rHb wild-type It has the sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0033] The invention also provides a method for producing hemoglobin by using the engineering yeast of saccharomyces cerevisiae.

[0034] In one embodiment, the method comprises culturing the engineered Saccharomyces cerevisiae in a heme synthesis medium; the heme synthesis medium contains yeast extract, peptone, glucose, glycine, succinic acid and citric acid.

[0035] In one embodiment, the method further comprises adding heme during culturing the Saccharomyces cerevisiae.

[0036] In one embodiment, the concentration of hemoglobin in the culture environment is 25 mg / L.

[0037] The invention also provides application of the brewer's yeast in producing hemoglobin or products containing hemoglobin.

[0038] Beneficial effects:

[0039] (1) The present invention obtains a mutant Hem1p that can relieve the feedback inhibition of heme on Hem1p in Saccharomyces cerevisiae by molecular docking of the key enzyme Hem1p in heme synthesis and heme, as well as further in vitro verification of the Hem1p mutant and in situ integration verification of the Saccharomyces cerevisiae genome. I213K Its ability to synthesize hemoglobin and ALA increased by 2.99 and 1.69 times compared with the control group CEN.PK2-1C.

[0040] (2) In order to further improve the heme supply in Saccharomyces cerevisiae and synthesize highly active human hemoglobin, Hem1p was integrated in situ into the Saccharomyces cerevisiae genome. I213K Based on the integration of multiple copies of the key intermediate enzyme HEM1 in heme synthesis I231K The heme production of the heme synthesis-enhanced strain H25 was 16.57 times higher than that of CEN.PK2-1C. When rHb was expressed in the H25 strain and CEN.PK2-1C supplemented with 25 mg / L heme, the production of rHb and the heme binding rate in H25 increased by 22.9% and 39.2% compared with those in CEN.PK2-1C. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1The effect of Hem1p alanine mutant on the feedback inhibition of heme in Saccharomyces cerevisiae. Among them, a is the verification of the expression of Hem1p in the BL21 (DE3) strain and the feedback inhibition of heme. B is the HEM1 mutation site identified by alanine scanning. 0.40 represents the binding mutation energy of the alanine virtual mutant, and 1-12 represents the mutant amino acid sites of subsequent experiments. C is the yield and SDS-PAGE analysis of Hem1p alanine mutant in Escherichia coli BL21 (DE3) strain. D is the verification of the feedback inhibition of heme on Hem1p alanine mutant.

[0042] Figure 2 : Effect of Hem1p saturation mutant on feedback inhibition of heme in Saccharomyces cerevisiae. Wherein, a is the HEM1 mutation site identified by virtual saturation mutation. 1.00 represents the binding mutation energy of the virtual saturation mutant. b is the yield and SDS-PAGE analysis of Hem1p saturation mutant in Escherichia coli BL21 (DE3) strain. c is the verification of feedback inhibition of heme on Hem1p saturation mutant. d is the yield, SDS-PAGE analysis and enzyme activity of Hem1p combination mutant in Escherichia coli BL21 (DE3) strain.

[0043] Figure 3 The construction and effect verification of the engineered strain of Saccharomyces cerevisiae that relieves the feedback inhibition of heme. Among them, a is the engineering strategy for relieving the feedback inhibition of heme on Hem1p. b is the production of ALA and heme of the engineered strain of Saccharomyces cerevisiae that relieves the feedback inhibition of heme.

[0044] Figure 4 Construction of a Saccharomyces cerevisiae strain with enhanced heme supply and synthesis of highly active human hemoglobin. Where a is the gene copy number and heme content of the HEM13 multi-copy integrated strain. b is the gene copy number of HEM13 and HEM1 I213k The gene copy number and ALA and heme production in the multi-copy integrated strain. c is the biomass of the HEM13 multi-copy integrated strain. d is the biomass of HEM13 and HEM1 I213k The biomass of the multi-copy integrated strain. e is the biomass of the strain synthesizing rHb in the Saccharomyces cerevisiae strain with enhanced heme supply. f is the yield and heme binding rate of rHb synthesized in the Saccharomyces cerevisiae strain with enhanced heme supply. DETAILED DESCRIPTION

[0045] Heme synthesis medium (by final concentration): 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 7.5 g / L glycine, 2.5 g / L succinic acid and 0.1 mM citric acid.

[0046] Molecular docking of Hem1p with heme: We constructed two Hem1p structures containing cofactors (pyridoxal phosphate) and substrates (succinyl-CoA and glycine), namely: (1) Hem1p-pyridoxal phosphate-glycine-succinyl-CoA; (2) Hem1p-glycine-succinyl-CoA. Subsequently, the CDOCKER module in Discovery Studio 2019 was used to perform semi-flexible molecular docking of these two Hem1p structures with heme. The main parameters of the CDOCKER module were set as follows: Pose Cluster Radius was 0.5, Maximum Bad Orientations was 2000, Orientation vdWEnergy Threshold was 800, and other parameters were set to default values. In order to evaluate the reliability of the molecular dynamics simulation, the docking morphology with the highest score was selected as the candidate based on the CDOCKER score (CDOCKER_ENERGY). Subsequently, the sites among the candidates were further screened by alanine scanning, and hotspot amino acid sites (BindingEnergies>0.1) were identified to eliminate the feedback inhibition of Hem1p by heme.

[0047] Determination of Hem1p enzyme activity: The enzyme activity of Hem1p was calculated by colorimetry. First, 6.0 μM Hem1p was incubated with different concentrations of heme (0.0, 1.0 and 10.0 μM) in a reaction mixture containing 50.0 μL of 1.0 M glycine, 50.0 μL of 0.2 M succinic acid, 5.0 μL of pyridoxal phosphate and 20.0 mM magnesium chloride, and the final volume was adjusted to 500.0 μL. The mixed reaction solution was incubated at 37°C for 10 minutes, and then 150.0 μL of 10.0% trichloroacetic acid was added to terminate the reaction. After centrifugation (7000 rpm, 5 minutes), 0.3 mL of the supernatant was taken and mixed with 0.4 mL of a neutralizing reagent (1.0 M sodium acetate, pH 4.6) and 35.0 μL of acetylacetone. The mixture was boiled for 15 minutes and cooled to room temperature. Subsequently, 0.2 mL of ALA reaction solution was added, which contained 42.0 mL of acetic acid, 8.0 mL of 70.0% perchloric acid, and 1.0 g of p-dimethylaminobenzaldehyde. The mixture was incubated at room temperature for 10 minutes and then read at A using a microplate reader (BioTek synergy H1). 554 The absorbance was measured at 4 °C to characterize the ALA content, thereby reflecting the catalytic ability of Hem1p.

[0048] Residual enzyme activity (%): the ratio of the enzyme activity of Hem1p or its mutants in a heme-containing environment to the enzyme activity in a heme-free environment; wherein the enzyme activity is determined by the aforementioned method.

[0049] Determination of ALA content by HPLC: The parameters of HPLC are as follows: the injection volume is 10 μL, and the chromatographic column is Agilent C-18 column (1.8 μm, 4.6 mm×150 mm). The sample is treated with o-phthalaldehyde (OPA) as a pre-column derivatization reagent. The mobile phase consists of solution A (36 mM sodium acetate buffer, containing 0.2 mL / L triethylamine, 5 mL / L tetrahydrofuran, pH 7.2) and solution B (36 mM sodium acetate, 40.0% methanol, 40.0% acetonitrile, pH 7.2). The column temperature is maintained at 40°C, the flow rate is set to 1.0 mL / min, and the detection wavelength is set to A. 338 .

[0050] Heme detection method: The Saccharomyces cerevisiae strain with enhanced heme supply was cultured in a heme synthesis medium (1.0% yeast extract, 2.0% peptone, 2.0% glucose, 0.75% glycine, 0.25% succinic acid and 0.1 mM citric acid) at 30°C for 48 hours (rotation speed 220 rpm). The cells were collected by centrifugation (7000 rpm) at 4°C and washed twice with deionized water. The OD 600 Cells with a value of 40 were resuspended in 500 μL of detection reagent (50.0% acetonitrile, 15.0% ammonia, and 35.0% deionized water) and precipitated using FastPrep-24 TM Cell lysis was performed using a bead beating and lysis system (MP Biomedicals).

[0051] The heme content was detected by liquid chromatography-mass spectrometry (Agilent G6400, USA). 1 μL of sample was loaded onto an Agilent ZORBAX Eclipse Plus C18 column (2.1×50 mm, 1.8 μm), and the sample was separated using an Agilent 1290 Infinity II LC system. Quantitative analysis was performed by an Agilent 6495C triple quadrupole mass spectrometer (Agilent, USA). Two mobile phases were selected for sample analysis at a flow rate of 0.4 mL / min, including mobile phase A (10 mM ammonium formate, 0.1% formic acid) and mobile phase B (0.1% formic acid dissolved in acetonitrile). The production of heme was monitored at a specific mass-to-charge ratio (m / z 616.2→557.2).

[0052] Heme binding rate detection method: The heme binding rate in rHb was determined by the pyridine heme method. First, a pyridine mixture was prepared at room temperature, including: 1.75 mL of pyridine, 0.75 mL of 1M sodium hydroxide. Further, 0.75 mL of 0.83 μM rHb, 0.25 mL of pyridine mixture and 2 mg of sodium bisulfite were mixed in 0.1 M PBS buffer (pH 7.4), the absorbance at 414 nm was detected and the binding ratio of heme (%) mol / mol hemoglobin was further determined:

[0053] ε 414 =342500M -1 cm -1 .

[0054] Copy number calculation method for multi-copy strains: In order to determine the copy number of the key gene for heme synthesis (HEM1 I213K and HEM13), we selected HEM1 I213K Saccharomyces cerevisiae strains with HEM13 and ALG9 genes were used as controls, and the housekeeping gene ALG9 was used as a housekeeping gene. RT-PCR primers for all genes were designed using Beacon Designer software. Total RNA was extracted using RNAprep Pure Plant Kit (Tiangen Biotechnology). cDNA was transcribed using PrimeScriptTM RT-PCR Kit (TaKaRa). RT-PCR experiments were performed using SYBR Premix Ex Taq Kit reaction mixture and LightCycler 480II real-time fluorescence quantitative PCR instrument, and 2 -ΔΔCt Methods Copy number analysis was performed.

[0055] Example 1 Interaction between Hem1p alanine mutant and heme

[0056] During the synthesis of heme, heme inhibits the synthesis of its precursor ALA, thereby controlling the level of heme in cells. In order to explore this regulatory mechanism, the leading peptide (2nd to 62nd residues at the N-terminus) that affects the soluble heterologous expression of Hem1p (Genbank accession number: NM_001180540.1) was removed, and 6 histidine tags were fused to the C-terminus of Hem1p to facilitate protein purification. At the same time, the maltose binding protein (MBP) carried by the pMAL-c5X vector itself was used to promote the active expression of Hem1p. The gene sequence of this protein is shown in SEQ ID NO.6. Both genes were co-expressed under the control of the tac promoter to construct the plasmid pMAL-Hem1p.wild-type . The recombinant plasmid pMAL-Hem1p wild-type Transformed into E. coli BL21 (DE3) strain and cultured in Luria-Bertani medium (100 mg / L ampicillin) at 37°C (220 rpm) until OD 600 When the value reached 0.6, the strain was induced with 0.25mM IPTG and cultured for another 4 hours at 28°C (220rpm). The cells were collected by centrifugation (7000rpm, 10 minutes) and resuspended in 50.0mM Tris-HCl buffer (pH 7.5). Subsequently, the cells were broken by ultrasonic treatment at 4°C for 20 minutes. The supernatant after disruption was centrifuged at 4°C for 10 minutes (7000rpm) and loaded onto a histidine-tagged affinity gravity column (Shanghai Dianchuang). Impurities were removed using 50.0mM imidazole (pH 7.5, 50mM Tris-HCl), and the target protein Hem1p was then eluted with 0.5M imidazole (pH7.5, 50mM Tris-HCl). Use Ultra-0.5 centrifugal filter (Merck KGaA) was used to reduce the imidazole concentration in the purified Hem1p solution to less than 50.0 mM. 10 μM heme was added to the 6.0 μM Hem1p pure enzyme at a final concentration, and the effect of heme on the Hem1p enzyme activity was verified by colorimetry as described in the above determination of Hem1p enzyme activity. The results showed that when 10 μM heme was added, the residual enzyme activity of Hem1p was only 6.1±1.2% (0.0447±0.034) of the enzyme activity of 6.0 μM Hem1p without heme. Figure 1 ), indicating that there may be a heme-binding region in the Hem1p structure.

[0057] Therefore, the CDOCKER module in Discovery Studio 2019 was used to perform semi-flexible molecular docking between the active region of Hem1p and heme, and 23 residues that may interact with heme were identified. These residues were further subjected to virtual alanine scanning, and 12 Hem1p mutation sites (T150A, I153A, Y183A, E206A, H209A, I213A, M257A, H284A, F365A, I448A, P450A and F451A sites) with significantly reduced binding mutation energy (>0.4 kcal / mol) were selected, and pMAL-Hem1p wild-type Similar to the construction method of , the tac promoter was used to control the co-expression of the corresponding Hem1p mutant gene and maltose binding protein, thereby constructing 12 corresponding recombinant plasmids. The recombinant plasmid carrying the Hem1p mutant gene (e.g., pMAL-Hem1p T150A) was transformed into E. coli BL21 (DE3). The verified correct strain was cloned into pMAL-Hem1p wild-type Expression and purification of Hem1p and the effect of heme on Hem1p wild-type The results showed that the five mutants (Hem1p I153A 、Hem1p Y183A 、Hem1p I213A 、Hem1p F365A and Hem1p P450A ) maintained a residual enzyme activity between 73.1±5.7% and 86.1±4.2%, demonstrating that these mutants significantly alleviated the feedback inhibition of Hem1p by heme.

[0058] Example 2 Interaction between Hem1p saturation mutant and heme

[0059] On the basis of Example 1, in order to further alleviate the feedback inhibition of heme on Hem1p, the five alanine mutants Hem1p screened in Example 1 were I153A 、Hem1p Y183A 、Hem1p I213A 、Hem1p F365A and Hem1p P450A Virtual saturation mutagenesis was performed, and 16 Hem1p mutants with significantly reduced binding mutation energy (>1.0 kcal / mol) were selected, namely I153G, I153S, I153K, Y183K, Y183G, Y183S, Y183V, H209K, H209G, I213K, I213R, F365Q, F365R, F365K, F450K and F450R. The corresponding 16 recombinant plasmids such as pMAL-Hem1p were constructed using the pMAL-c5X vector according to the method of Example 1. I153G et al., expressed and purified the corresponding Hem1p mutant enzyme in BL21(DE3).

[0060] As in Example 1, pMAL-Hem1p wild-type Expression and purification of Hem1p and the effect of heme on Hem1p wild-type The results showed that when 10 μM heme was added, the four mutants (Hem1p I153G 、Hem1p Y183G 、Hem1p I213K and Hem1p I213R ) with residual enzyme activity ranging from 86.3±1.6% to 92.9±2.1% ( Figure 2), indicating that these saturation mutants further enhance their ability to resist feedback inhibition by heme.

[0061] Among all the forward mutants, four key sites with better performance (Hem1p I153G 、Hem1p Y183G 、Hem1p I213K and Hem1p F365A ) mutations were combined to obtain mutant Hem1p I213K / Y183G 、Hem1p I213K / I153G 、Hem1p I213K / Y183G / I153G 、Hem1p I213K / Y183G / F365A and Hem1p I213K / Y183G / I153G / F365A As in Example 1, pMAL-Hem1p wild-type Expression and purification of Hem1p and the effect of heme on Hem1p wild-type The corresponding Hem1p mutant enzyme was obtained and its function was verified by the validation method. Figure 2 D shows the residual enzyme activity of the combined mutants compared with Hem1p wild-type Significantly decreased (<13.0%).

[0062] Example 3 In situ integration of Hem1p mutants into the Saccharomyces cerevisiae genome and verification of integration effect

[0063] In order to construct a strain that relieves heme feedback inhibition in Saccharomyces cerevisiae, the residual enzyme activity of Example 1 and Example 2 was higher (compared to Hem1p wild-type Six Hem1p variants retaining the leader peptide (residual enzyme activity> 80.0%) I153G 、Hem1p Y183A 、Hem1p Y183G 、Hem1p I213R 、Hem1p I213K and Hem1p F365A Hem1 was integrated into the CEN.PK2-1C genome wild-type In situ integration of Hem1p I153G The specific steps are to use PCR to amplify the HEM1 of the CEN.PK2-1C genome. wild-type The 800 bp fragments on both sides of the coding region were used as homology arms, and the two homology arms were fused to the mScarlet coding gene and Hem1p gene respectively using the Gibson assembly method. I153G The two fusion fragments Up-mScarlet-Down and Up-Hem1p were constructed on both sides of the coding gene. I153G-Down. Using CRISPR / Cas9 technology, 1 mg of Up-mScarlet-Down fusion fragment was used to replace Hem1 in the CEN.PK2-1C genome by lithium acetate transformation. wild-type , thus obtaining the integrated strain H-mScarlet. Subsequently, 1 mg of Up-Hem1p I153G -Down fusion fragment replaces the mScarlet region of the integrated strain H-mScarlet, thereby obtaining in situ integrated Hem1p I153G strain H1.

[0064] In order to investigate the effect of intracellular heme on the Hem1p mutant, two other key enzymes for heme synthesis, Hem3p (Gene ID: 851322; Genbank accession number: NM_001180265.1) and Hem13p (Gene ID: 851322; Genbank accession number: NM_001180265.1), were co-expressed in the H1-H6 strain and the control group (CEN.PK2-1C strain). ID: 851614; Genbank accession number: NM_001180352.1), the specific steps are: using Gibson assembly to fuse the coding genes of Hem3p and Hem13p respectively into the downstream of TEF1 promoter and GPD promoter of pY26 vector, thereby constructing the co-expression plasmid pY26-Hem3p-Hem13p, and the plasmid pY26-Hem3p-Hem13p was transformed into H1-H6 strain and CEN.PK2-1C strain respectively by lithium acetate transformation method, thereby constructing strains H1-3p-13p ~ strains H6-3p-13p and strain CEN-3p-13p. The strains H1-3p-13p, H2-3p-13p, H3-3p-13p, H4-3p-13p, H5-3p-13, H6-3p-13p and strain CEN-3p-13p were cultured in heme synthesis medium at 30°C and 220rpm for 48 hours, and the heme and ALA contents in the fermentation broth were detected. The results showed that among the six Hem1p mutants, the ALA (59.45±5.53mg / L) and heme (1.48±0.34mg / L) production of strain H5-3p-13 were increased by 1.69 times and 2.99 times, respectively, compared with the control group. Figure 3 ). This result indicates that Hem1p I213K The mutant can enhance the synthesis of ALA and the supply of heme in the body.

[0065] Example 4: Multi-copy integration and expression of key enzymes for heme synthesis promotes the synthesis of active human hemoglobin

[0066] In order to increase the supply of heme in Saccharomyces cerevisiae, based on the H5 strain obtained in Example 3, the key enzyme Hem1p in heme synthesis was overexpressed. I213K (The gene sequence encoded is as shown in SEQ ID NO.8) and Hem13p (Genbank accession number: NM_001180352.1). The specific steps are to use PCR to amplify 800bp fragments on both sides of the rDNA site of the CEN.PK2-1C genome as homology arms, and use the Gibson assembly method to fuse these two homology arms to both sides of the HEM13 coding gene, thereby constructing the fusion fragment Up-HEM13-Down. Using CRISPR / Cas9 technology, 1mg of the Up-HEM13-Down fusion fragment was integrated into the rDNA site of the CEN.PK2-1C genome by lithium acetate transformation, and the gene copy number of the multi-copy strain was determined by RT-PCR to construct a strain expressing one copy of Hem1p I213K The strains expressing the Hem13p gene and 1 to 13 copies of the Hem13p gene (Genbank accession number: NM_001180352.1) were named H7 to H17, respectively.

[0067] The strains H7 to H17 were cultured in heme synthesis medium at 30°C for 48 hours (rotation speed 220 rpm) and the OD per unit volume was 600 Cells with a value of 40 were resuspended in 500 μL of detection reagent (50.0% acetonitrile, 15.0% ammonia, and 35.0% deionized water) and precipitated using FastPrep-24 TM Cells were lysed using a bead mill and lysis system (MP Biomedicals), and the supernatant was collected after centrifugation. The heme synthesis capacity of the strains was tested by GC-MS. The results showed that strain H13 (containing 8 copies of the HEM13 gene) had the strongest heme synthesis capacity, reaching 0.16±0.02 mg / L (this concentration is calculated based on the original unit volume). Furthermore, multiple copies of HEM1 were integrated into the Delta locus of the H13 strain. I213K The copy numbers of these strains were determined by RT-PCR, and clones containing 8 copies of the Hem13p gene (Genbank accession number: NM_001180352.1) and 1 to 11 copies of Hem1p I213K The strains with the same gene were named as strains H18 to H26.

[0068] The strains H18 to H26 were cultured in heme synthesis medium at 30°C for 48 hours (rotation speed 220 rpm), and the heme synthesis ability of these strains was detected by GC-MS. Finally, the H25 strain (9 copies of HEM1 I213Kgene) increased heme synthesis by 16.57 times compared with the control group CEN.PK2-1C, reaching 0.39±0.02mg / L ( Figure 4 ). Meanwhile, the enhancement of heme supply did not significantly affect the growth of H7-H26 strains.

[0069] Finally, the human hemoglobin encoding gene (nucleotide sequence as shown in SEQ ID NO.3 and SEQ ID NO.4) was cloned into the downstream of the GAL1-10 promoter of the inducible plasmid pESC-Ura by Gibson assembly, and the corresponding recombinant plasmid pESC-rHb was constructed, and the recombinant plasmid was transformed into the H25 strain and CEN.PK2-1C strain by lithium acetate transformation. After the H25 strain was cultured in heme synthesis medium at 30°C for 16 hours (speed 220rpm), 20mg / L galactose was added to induce the expression of human hemoglobin, and after further culture at 30°C for 48 hours (speed 220rpm), the cells were broken at 4°C by a high-pressure homogenizer (UnionBiotech), and the supernatant was collected by centrifugation at 4°C for 10 minutes (7000rpm), and the supernatant was added to the affinity gravity column of the His tag, and rHb was eluted with 0.5M imidazole in PBS buffer (pH 7.4). The protein concentration was determined using a Bradford protein assay kit (Beyotime Biotech), and the heme binding rate in rHb was determined using the pyridinium heme method. The results showed that compared with the control strain CEN.PK2-1C with 25 mg / L heme, the rHb production and heme binding rate in the H25 strain increased by 22.9% and 39.2%, reaching 15.70±0.47 mg / L and 43.73±3.41% mol heme / mol rHb, respectively, which were 7.19 times and 1.84 times that of CEN.PK2-1C without heme. Therefore, without the addition of heme, the application of the H25 strain with enhanced heme supply can achieve a more efficient and economical synthesis of highly active rHb.

[0070] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A mutant capable of relieving the feedback inhibition of 5-aminolevulinic acid synthase by heme, characterized in that: Based on the starting sequence, there are one or more mutations as follows: (a) mutating the isoleucine at position 153 to alanine or glycine; (b) mutating tyrosine at position 183 to alanine or glycine; (c) mutating the isoleucine at position 213 to alanine, lysine or arginine; (d) mutating phenylalanine at position 365 to alanine; (e) The proline at position 450 was mutated to alanine.

2. The mutant according to claim 1, characterized in that The starting sequence is shown as SEQ ID NO.

1.

3. A gene encoding the mutant according to claim 1.

4. A recombinant microorganism, characterized in that Expressing the amino acid sequence shown in (a) or (b): (a) the mutant according to claim 1 or 2; (b) Based on the sequence of (a), the sequence of amino acids 2 to 62 is removed.

5. The recombinant microorganism according to claim 4, characterized in that The microorganism is Saccharomyces cerevisiae, including but not limited to Saccharomyces cerevisiae CEN.PK2-1C.

6. An engineered strain of Saccharomyces cerevisiae, characterized in that: The HEM1 gene is replaced in situ on the genome with a gene encoding the mutant according to claim 1 or 2.

7. The engineered yeast of claim 6, characterized in that: The gene Hem3p and the gene Hem13p were also overexpressed.

8. The engineered yeast of claim 6, characterized in that: The engineered yeast of Saccharomyces cerevisiae has multiple copies of the HEM13 gene integrated into the rDNA site and / or multiple copies of the HEM1 gene integrated into the Delta site. I213K Gene: HEM1 I213K The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

9. The engineered yeast of claim 7 or 8, characterized in that: pESC is used as an expression vector to express human hemoglobin; the human hemoglobin rHb wild-type The sequences are shown in SEQ ID NO.3 and SEQ ID NO.

4.

10. Use of the mutant according to claim 1 or 2, or the recombinant microorganism according to claim 4 or 5, or the Saccharomyces cerevisiae according to any one of claims 6 to 9 in producing hemoglobin or a product containing hemoglobin.

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