Saccharomyces cerevisiae for relieving hemoglobin feedback inhibition and strengthening hemoglobin supply and application thereof

Abstract

The application discloses a Saccharomyces cerevisiae for relieving heme feedback inhibition and strengthening heme supply and application, and belongs to the technical field of genetic engineering. The application constructs a mutant of 5-aminoacetylpropionic acid synthetase which can relieve heme, and the yield of 5-aminoacetylpropionic acid and heme of the strain H5 constructed by integrating the mutant in situ in Saccharomyces cerevisiae is 1.69 and 2.99 times higher than that of the control strain CEN.PK2-1C respectively. Further, HEM1 I213K and HEM13 are integrated in multiple copies on the basis of the H5 strain, and the heme synthesis of the heme-strengthening strain H25 obtained is 16.57 times higher than that of CEN.PK2-1C, and the yield of the synthesized rHb and the heme combination rate are 22.9% and 39.2% higher than those of CEN.PK2-1C with the addition of 25 mg / L heme, which is beneficial to realize heme supply strengthening and synthesis of human hemoglobin with high activity.

Description

Technical Field

[0001] This invention relates to a brewing yeast that relieves heme feedback inhibition and enhances heme supply, and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] Human hemoglobin, a functional protein with heme as a cofactor, possesses the ability to store and transport oxygen, thus enabling its application in the construction of artificial oxygen carriers. Currently, using the food-safe host, *Saccharomyces cerevisiae*, as a host for the synthesis of recombinant human hemoglobin (rHb) offers advantages such as unrestricted supply and avoidance of pathogen contamination. However, due to the cytotoxicity of heme, the supply of intracellular heme in natural *Saccharomyces cerevisiae* is strictly regulated. Therefore, the synthesis of physiologically active human hemoglobin in *Saccharomyces cerevisiae* requires enhancing the intracellular heme supply level of the host.

[0003] Although exogenous addition of 5-aminolevulinic acid (ALA) or heme can enhance the expression of highly active hemoglobin, these strategies are not very effective. Moderately increasing heme synthesis in the microbial host can overcome these limitations. While high-titer heme-synthesizing strains can be obtained by enhancing multiple rate-limiting enzymes in the heme synthesis pathway, and by alleviating the steric hindrance of heme synthases (Hem14p and Hem15p), the activity of key rate-limiting enzymes in *Saccharomyces cerevisiae* can be increased, resulting in bovine hemoglobin at 18.0% mol heme / mol. However, to further construct a heme-enhanced microbial platform for the efficient synthesis of highly active rHb, several issues still need to be addressed. For example, the feedback inhibition of the key synthase 5-aminolevulinic acid synthase (Hem1p) by heme in *Saccharomyces cerevisiae* exacerbates unnecessary waste of cellular resources during heme supply enhancement, thus adversely affecting subsequent protein synthesis.

[0004] Therefore, in order to improve the supply level of heme in Saccharomyces cerevisiae while conserving cellular resources as much as possible, it is an urgent problem to solve how to alleviate the feedback inhibition of the key synthase Hem1p by heme. Summary of the Invention

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

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

[0007] (b) Mutate the tyrosine at position 183 to either alanine or glycine;

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

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

[0010] (e) Mutate proline at position 450 to alanine.

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

[0012] In one embodiment, the Hem1p alanine mutant capable of alleviating 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 feedback inhibition of heme includes: Hem1p I153G Hem1p Y183G Hem1p I213K and Hem1p I213R .

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

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

[0016] The present invention also provides recombinant microbial cells 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 of Hem1p (61 amino acid residues from amino acid position 2 to 62 at the N-terminus) 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 the *Escherichia coli* is *Escherichia coli* BL21(DE3).

[0022] This invention also provides an engineered Saccharomyces cerevisiae strain that relieves the feedback inhibition of Hem1p by heme, wherein the engineered Saccharomyces cerevisiae strain has the gene HEM1 replaced in situ with HEM1 in the genome. I213K .

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

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

[0025] In one embodiment, the engineered Saccharomyces cerevisiae also overexpressed 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 Saccharomyces cerevisiae integrates multiple copies of the HEM13 gene at the rDNA site and / or multiple copies of HEM1 at the Delta site. I213K Gene.

[0027] In one embodiment, the nucleotide sequence of the HEM13 gene is shown in SEQ ID NO. 5; HEM1 I213K The nucleotide sequence of the gene is shown in SEQ ID NO.8.

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

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

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

[0031] In one embodiment, the brewer's yeast uses pESC as an expression vector to express the human hemoglobin gene.

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

[0033] The present invention also provides a method for producing hemoglobin using the engineered strain of *Saccharomyces cerevisiae*.

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

[0035] In one embodiment, the method further includes adding heme during the cultivation of the brewing yeast.

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

[0037] The present invention also provides the application of the brewing yeast in the production of hemoglobin or hemoglobin-containing products.

[0038] Beneficial effects:

[0039] (1) This invention obtained a mutant Hem1p that can relieve the feedback inhibition of Hem1p by heme in Saccharomyces cerevisiae by heme through molecular docking of Hem1p, a key enzyme 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 heme and ALA was 2.99 times and 1.69 times higher than that of the control group CEN.PK2-1C, respectively.

[0040] (2) In order to further improve the supply of heme in Saccharomyces cerevisiae and the synthesis of highly active human hemoglobin, Hem1p was integrated in situ into the Saccharomyces cerevisiae genome. I213K Based on this, multiple copies of the key intermediate enzyme system HEM1 for heme synthesis were integrated. I231K Compared to CEN.PK2-1C, the heme synthesis-enhanced strain H25, obtained by comparing it with HEM13, produced 16.57 times more heme. When rHb was expressed in strain H25 and CEN.PK2-1C supplemented with 25 mg / L heme, the rHb production and heme binding rate in H25 increased by 22.9% and 39.2%, respectively, compared to CEN.PK2-1C. Attached Figure Description

[0041] Figure 1This study investigated the effect of the Hem1p alanine mutant on feedback inhibition of heme in *Saccharomyces cerevisiae*. In the figures, a) validates the expression of Hem1p in strain BL21(DE3) and the feedback inhibition of heme. b) shows the HEM1 mutation site identified by alanine scanning. 0.40 represents the binding mutagenicity of the alanine virtual mutant, and 1-12 represent the mutated amino acid sites in subsequent experiments. c) shows the yield of the Hem1p alanine mutant in *Escherichia coli* strain BL21(DE3) and SDS-PAGE analysis. d) validates the feedback inhibition of heme by the Hem1p alanine mutant.

[0042] Figure 2 This study investigates the effect of the Hem1p saturated mutant in *Saccharomyces cerevisiae* on feedback inhibition of heme. In the figures, a represents the HEM1 mutation site identified through virtual saturation mutation. 1.00 represents the binding mutagenicity of the virtual saturated mutant. b shows the yield and SDS-PAGE analysis of the Hem1p saturated mutant in *Escherichia coli* BL21(DE3) strain. c validates the feedback inhibition of heme by the Hem1p saturated mutant. d shows the yield, SDS-PAGE analysis, and enzyme activity of the Hem1p combinatorial mutant in *Escherichia coli* BL21(DE3) strain.

[0043] Figure 3 To construct and validate an engineered Saccharomyces cerevisiae strain that relieves heme feedback inhibition. Here, a represents the engineering strategy for relieving heme feedback inhibition on Hem1p. b represents the ALA and heme yields of the engineered Saccharomyces cerevisiae strain that relieved heme feedback inhibition.

[0044] Figure 4 Construction of a heme-enhanced Saccharomyces cerevisiae strain and synthesis of highly active human hemoglobin. In this study, a represents the gene copy number and heme content of the HEM13 multicopy integration strain. b represents the gene copy number and heme content of HEM13 and HEM1... I213k Gene copy number and ALA and heme production in the multicopy-integrated strain. c represents the biomass of the HEM13 multicopy-integrated strain. d represents the biomass of HEM13 and HEM1. I213k Biomass of multicopy-integrating strains. e represents the biomass of rHb synthesized in heme-enhanced Saccharomyces cerevisiae strains. f represents the yield of rHb synthesized and the heme binding rate in heme-enhanced Saccharomyces cerevisiae strains. Detailed Implementation

[0045] Heme synthesis medium (at 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 a cofactor (pyridoxal phosphate) and substrates (succinyl-CoA and glycine), namely: (1) Hem1p-pyridoxal phosphate-glycine-succinyl-CoA; (2) Hem1p-glycine-succinyl-CoA. Subsequently, using the CDOCKER module in Discovery Studio 2019, we performed 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. 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 in the candidate samples were further screened by alanine scanning to identify hot amino acid sites (BindingEnergies>0.1) in order to eliminate the feedback inhibition of Hem1p by heme.

[0047] Determination of Hem1p enzyme activity: The enzyme activity of Hem1p was calculated by colorimetric method. 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, with the final volume adjusted to 500.0 μL. The reaction mixture was incubated at 37 °C for 10 min, and then 150.0 μL of 10.0% trichloroacetic acid was added to terminate the reaction. After centrifugation (7000 rpm, 5 min), 0.3 mL of the supernatant was mixed with 0.4 mL of neutralizing reagent (1.0 M sodium acetate, pH 4.6) and 35.0 μL of acetylacetone. The mixture was boiled for 15 min 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 the microplate readings were obtained using a BioTek Synergy H1 microplate reader. 554 The absorbance was measured 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 mutant 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 high performance liquid chromatography (HPLC): The HPLC parameters were as follows: injection volume was 10 μL, and an Agilent C-18 column (1.8 μm, 4.6 mm × 150 mm) was used. Orthophthalaldehyde (OPA) was used as the pre-column derivatization reagent to treat the sample. The mobile phase consisted of solution A (36 mM sodium acetate buffer, containing 0.2 mL / L triethylamine and 5 mL / L tetrahydrofuran, pH 7.2) and solution B (36 mM sodium acetate, 40.0% methanol, and 40.0% acetonitrile, pH 7.2). The column temperature was maintained at 40 °C, the flow rate was set to 1.0 mL / min, and the detection wavelength was set to A. 338 .

[0050] Heme detection method: A heme-enhanced *Saccharomyces cerevisiae* strain was cultured at 30°C for 48 hours (220 rpm) in 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). Cells were collected by centrifugation (7000 rpm) at 4°C and washed twice with deionized water. OD... 600 Cells with a value of 40 were resuspended in 500 μL of assay reagent (50.0% acetonitrile, 15.0% ammonia, and 35.0% deionized water) and tested using FastPrep-24. TM Cell lysis is performed using a bead milling and lysis system (MP Biomedicals).

[0051] Heme content was determined 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 sample separation was performed using an Agilent 1290 Infinity II LC system. Quantitative analysis was performed using 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: mobile phase A (10 mM ammonium formate, 0.1% formic acid) and mobile phase B (0.1% formic acid dissolved in acetonitrile). Heme yield was monitored at specific mass-to-charge ratios (m / z 616.2→557.2).

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

[0053] ε 414 =342500M -1 cm -1 .

[0054] Methods for calculating the copy number of multicopy strains: To determine the key gene for heme synthesis (HEM1) in multicopy integrated yeast strains. I213K Regarding the copy number of HEM1 and HEM13, we selected HEM1 including single copies. I213K A *Saccharomyces cerevisiae* strain with the HEM13 gene was used as a control, while the ALG9 gene was used as a housekeeping gene. RT-PCR primers for all genes were designed using Beacon Designer software. Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen Biotech). cDNA transcription was performed using the PrimeScript™ RT-PCR Kit (TaKaRa). RT-PCR experiments were performed using the SYBR Premix Ex Taq Kit reaction mixture and a LightCycler 480II real-time quantitative PCR instrument, and the results were analyzed using 2... -ΔΔCt The method included copy number analysis.

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

[0056] Heme inhibits the synthesis of its precursor ALA during heme synthesis, thereby controlling intracellular heme levels. To investigate this regulatory mechanism, the leader peptide (N-terminal residues 2 to 62) affecting the soluble heterologous expression of Hem1p (Genbank accession number: NM_001180540.1) was removed, and a 6-histidine tag was fused to the C-terminus of Hem1p to facilitate protein purification. Simultaneously, the maltose-binding protein (MBP) carried by the pMAL-c5X vector 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, thus constructing the plasmid pMAL-Hem1p.wild-type The recombinant plasmid pMAL-Hem1p wild-type Transformed into *Escherichia coli* BL21(DE3) strain. Cultured in Luria-Bertani medium (100 mg / L ampicillin) at 37°C (220 rpm) until OD... 600 When the pH reached 0.6, the strain was induced with 0.25 mM IPTG and cultured at 28°C for 4 hours (220 rpm). Cells were collected by centrifugation (7000 rpm, 10 min) and resuspended in 50.0 mM Tris-HCl buffer (pH 7.5). Cells were then lysed by sonication at 4°C for 20 min. The lysed supernatant was centrifuged at 4°C for 10 min (7000 rpm) and loaded onto a histidine-tagged affinity gravity column (Shanghai Dianchuang). Impurities were removed using 50.0 mM imidazole (pH 7.5, 50 mM Tris-HCl), followed by elution of the target protein Hem1p with 0.5 M imidazole (pH 7.5, 50 mM Tris-HCl). An Ultra-0.5 centrifugal filter (Merck KGaA) was used to reduce the imidazole concentration in the purified Hem1p solution to below 50.0 mM. 10 μM heme was added to 6.0 μM of purified Hem1p enzyme at a final concentration, and the effect of heme on Hem1p enzyme activity was verified by colorimetric assay as described in the previous section on Hem1p enzyme activity determination. The results showed that, compared to the enzyme activity of 6.0 μM Hem1p without heme, the residual enzyme activity of Hem1p after adding 10 μM heme was only 6.1 ± 1.2% ( ). Figure 1 This indicates that a heme-specific binding region may exist in the Hem1p structure.

[0057] Therefore, semi-flexible molecular docking of the active region of Hem1p with heme was performed using the CDOCKER module in Discovery Studio 2019, and 23 residues that may interact with heme were identified. A virtual alanine scan was then performed on these residues, and 12 Hem1p mutation sites (T150A, I153A, Y183A, E206A, H209A, I213A, M257A, H284A, F365A, I448A, P450A, and F451A) that showed significant binding reduction (>0.4 kcal / mol) were selected, along with pMAL-Hem1p. wild-type The construction methods are similar, utilizing the tac promoter to control the co-expression of the corresponding Hem1p mutant gene and maltose-binding protein, thereby constructing 12 corresponding recombinant plasmids. Recombinant plasmids carrying the Hem1p mutant gene (e.g., pMAL-Hem1p) were then used. T150ATransformed into Escherichia coli BL21(DE3). The verified strain was then transferred as described above (pMAL-Hem1p). wild-type Expression, purification and heme effects of Hem1p wild-type The verification method was used to obtain and functionally verify the corresponding Hem1p mutant enzymes. The results showed that five mutants (Hem1p...) I153A Hem1p Y183A Hem1p I213A Hem1p F365A and Hem1p P450A The residual enzyme activity remained 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 saturated mutant and heme

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

[0060] As in Example 1, pMAL-Hem1p wild-type Expression, purification and heme effects of Hem1p wild-type The verification method was used to obtain and functionally verify the corresponding Hem1p mutant enzymes. The results showed that when 10 μM heme was added, the four mutants (Hem1p...)... I153G Hem1p Y183G Hem1p I213K and Hem1p I213R The residual enzyme activity remained between 86.3±1.6% and 92.9±2.1%. Figure 2This indicates that these saturated mutants further enhance their ability to resist feedback inhibition of heme.

[0061] Among all positive mutants, four key sites (Hem1p) showed better performance. I153G Hem1p Y183G Hem1p I213K and Hem1p F365A Combining mutations were performed on the mutations to obtain the 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, purification and heme effects of Hem1p wild-type The verification method was used to obtain and functionally verify the corresponding Hem1p mutant enzyme. The results are as follows: Figure 2 As shown in D, the residual enzyme activity of the combined mutant compared to Hem1p wild-type Significantly reduced (<13.0%).

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

[0063] To construct a *Saccharomyces cerevisiae* strain that relieves heme feedback inhibition, strains with higher residual enzyme activity (compared to Hem1p) from Examples 1 and 2 were used. wild-type Six Hem1p variants that retain the leader peptide (with residual enzyme activity >80.0%) I153G Hem1p Y183A Hem1p Y183G Hem1p I213R Hem1p I213K and Hem1p F365A Hem1 cells were in situ integrated into the CEN.PK2-1C genome. wild-type H1-H6 strains were constructed at the site. Hem1p was integrated in situ. I153G Taking strain H1 as an example, the specific steps are as follows: PCR amplification of the HEM1 genome of CEN.PK2-1C. wild-type 800bp segments flanking the coding region were used as homologous arms, and these two homologous arms were fused into the mScarlet coding gene and Hem1p gene, respectively, using the Gibson assembly method. I153G Two fusion fragments, Up-mScarlet-Down and Up-Hem1p, were constructed by flanking the coding gene. I153G-Down. Using CRISPR / Cas9 technology, 1 mg of the Up-mScarlet-Down fusion fragment was used to replace the Hem1 fragment in the CEN.PK2-1C genome via lithium acetate conversion. wild-type This yielded the integrated strain H-mScarlet. Subsequently, 1 mg of Up-Hem1p was administered using the same method. I153G -The down fusion fragment replaces the mScarlet region of the integrated strain H-mScarlet, thereby obtaining in situ integrated Hem1p I153G strain H1.

[0064] To facilitate the investigation of 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 strains H1-H6 and the control group (CEN.PK2-1C strain). ID:851614; Genbank accession number: NM_001180352.1), the specific steps are as follows: using Gibson assembly, the coding genes of Hem3p and Hem13p were fused downstream of the TEF1 promoter and GPD promoter of the pY26 vector, respectively, to construct the co-expression plasmid pY26-Hem3p-Hem13p, and the plasmid pY26-Hem3p-Hem13p was transformed into strains H1-H6 and CEN.PK2-1C, respectively, using lithium acetate transformation, to construct strains H1-3p-13p, H6-3p-13p and CEN-3p-13p. Strains H1-3p-13p, H2-3p-13p, H3-3p-13p, H4-3p-13p, H5-3p-13p, H6-3p-13p, and CEN-3p-13p were cultured in heme synthesis medium at 30℃ and 220 rpm for 48 hours. The heme and ALA contents in the fermentation broth were then measured. The results showed that among the six Hem1p mutants, strain H5-3p-13 exhibited 1.69-fold (59.45±5.53 mg / L) and 2.99-fold (1.48±0.34 mg / L) higher ALA (1.69 ± 5.53 mg / L) and heme (1.48 ± 0.34 mg / L) yields compared to the control group. Figure 3 This result indicates that Hem1p I213K The mutant can enhance ALA synthesis and the supply of heme in the body.

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

[0066] To increase the supply of heme in Saccharomyces cerevisiae, the key enzyme Hem1p for heme synthesis was overexpressed based on the H5 strain obtained in Example 3. I213K (The encoded gene sequence is shown in SEQ ID NO. 8) and Hem13p (Genbank accession number: NM_001180352.1). The specific steps involved amplifying 800bp fragments flanking the rDNA site of the CEN.PK2-1C genome using PCR as homologous arms. These two homologous arms were then fused to both sides of the HEM13 encoding gene using the Gibson assembly method, thus constructing the fusion fragment Up-HEM13-Down. Using CRISPR / Cas9 technology, 1 mg of the Up-HEM13-Down fusion fragment was integrated into the rDNA site of the CEN.PK2-1C genome via lithium acetate conversion. The copy number of the multi-copy strain was determined using RT-PCR, and a strain expressing one copy of Hem1p was constructed. I213K The strains containing the gene and 1 to 13 copies of the Hem13p gene (Genbank accession number: NM_001180352.1) were named H7 to H17, respectively.

[0067] Strains H7–H17 were cultured in heme synthesis medium at 30°C for 48 hours (rotation speed 220 rpm), and the OD per unit volume was measured. 600 Cells with a value of 40 were resuspended in 500 μL of assay reagent (50.0% acetonitrile, 15.0% ammonia, and 35.0% deionized water) and tested using FastPrep-24. TM Cell lysis was performed using a bead milling and lysis system (MP Biomedicals). After centrifugation, the supernatant was collected, and the intracellular heme synthesis capacity of the strains was detected 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 was calculated based on the original unit volume). Further investigation revealed that multiple copies of the HEM1 gene were integrated into the Delta site of strain H13. I213K Genes were analyzed, and the copy number of these strains was determined using RT-PCR. Genes integrating 8 copies of the Hem13p gene (Genbank accession number: NM_001180352.1) and 1–11 copies of Hem1p were constructed. I213K The strains containing the gene were named strains H18 to H26, respectively.

[0068] Strains H18–H26 were cultured in heme synthesis medium at 30°C for 48 hours (220 rpm). The heme synthesis capacity of these strains was detected by GC-MS. Finally, strain H25 (9 copies of HEM1) was identified as the most effective heme synthesizer. I213KThe heme synthesis of the gene was 16.57 times higher than that of the control group CEN.PK2-1C, reaching 0.39±0.02 mg / L. Figure 4 Meanwhile, the increased supply of heme did not significantly affect the growth of strain H7-H26.

[0069] Finally, the human hemoglobin encoding gene (nucleotide sequence shown in SEQ ID NO.3 and SEQ ID NO.4) was cloned downstream of the GAL1-10 promoter of the inducible plasmid pESC-Ura via Gibson assembly, constructing the corresponding recombinant plasmid pESC-rHb. This recombinant plasmid was then transformed into strains H25 and CEN.PK2-1C using lithium acetate conversion. After culturing strain H25 in heme synthesis medium at 30°C for 16 hours (220 rpm), 20 mg / L galactose was added to induce human hemoglobin expression. The cells were then cultured at 30°C for another 48 hours (220 rpm). Cells were then homogenized at 4°C using a high-pressure homogenizer (UnionBiotech), and the supernatant was collected by centrifugation at 4°C for 10 minutes (7000 rpm). The supernatant was added to a His-tagged affinity gravity column, and rHb was eluted with 0.5 M imidazole-buffered PBS (pH 7.4). Protein concentration was determined using the Bradford Protein Assay Kit (Beyotime Biotech), and the heme binding rate in rHb was determined using the pyridine heme method. The results showed that compared to the control strain CEN.PK2-1C supplemented with 25 mg / L heme, the rHb yield and heme binding rate in strain H25 were increased by 22.9% and 39.2%, respectively, reaching 15.70 ± 0.47 mg / L and 43.73 ± 3.41% mol heme / mol rHb, respectively. These figures were 7.19 times and 1.84 times higher than those of CEN.PK2-1C without heme supplementation. Therefore, the H25 strain with enhanced heme supply can achieve more efficient and economical synthesis of highly active rHb without the addition of heme.

[0070] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by 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 amino acid sequence shown in SEQ ID NO.1, amino acids 2 to 62 from the N-terminus were truncated, and any of the following mutations were performed: (a) Mutate isoleucine at position 153 to alanine or glycine; (b) Mutate tyrosine at position 183 to alanine or glycine; (c) Mutate isoleucine at position 213 to alanine, lysine, or arginine; (d) Mutate phenylalanine at position 365 to alanine; (e) Mutate proline at position 450 to alanine.

2. The gene encoding the mutant of claim 1.

3. A recombinant brewing yeast, characterized in that, The mutant of claim 1 was expressed in Saccharomyces cerevisiae CEN.PK2-1C.

4. The recombinant brewing yeast according to claim 3, characterized in that, Genes were added to the genome of Saccharomyces cerevisiae CEN.PK2-1C. HEM1 The gene encoding the mutant of claim 1 is replaced in situ.

5. The recombinant brewing yeast according to claim 4, characterized in that, The genes Hem3p and Hem13p were also overexpressed; the nucleotide sequence of the Hem3p gene is shown in NCBI Gene ID: 851322; the nucleotide sequence of the Hem13p gene is shown in NCBI Gene ID: 851614.

6. The recombinant brewing yeast according to claim 5, characterized in that, Integration of 1 to 11 copies at the Delta site HEM1 I213K Genes; the stated HEM1 I213K The nucleotide sequence of the gene is shown in SEQ ID NO.

8.

7. The recombinant brewing yeast according to claim 6, characterized in that, The recombinant Saccharomyces cerevisiae integrates 1 to 13 copies of its rDNA at the specified site. HEM13 Genes; the stated HEM13 The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

8. The recombinant brewing yeast according to any one of claims 5 to 7, characterized in that, Human hemoglobin was expressed using pESC as an expression vector; the human hemoglobin rHb wild-type The nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.

4.

9. The use of the mutant of claim 1, or the recombinant brewer's yeast of any one of claims 3 to 8, in the production of hemoglobin or hemoglobin-containing products.

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