Saccharomyces cerevisiae engineering bacterium for producing heme-binding protein as well as construction method and application of saccharomyces cerevisiae engineering bacterium

Abstract

The invention discloses a saccharomyces cerevisiae engineering bacterium for producing heme-binding protein as well as a construction method and application of the saccharomyces cerevisiae engineering bacterium. The construction method comprises the following steps: (1) by taking Saccharomyces cerevisiae CEN.PK2-1C as an original strain, optimizing a metabolic pathway, enhancing the supply of cytoplasm alpha-ketoglutaric acid (alpha-KG) and succinyl coenzyme A, and realizing the efficient accumulation of 5-aminolevulinic acid (ALA); (2) regulating and controlling the heme synthesis gene cluster in a time-sharing manner, and cooperatively expressing heme binding protein, so as to improve the assembly efficiency of heme (Heme); and (3) a secretion system is optimized, the secretion bottleneck of eukaryotic protein is broken through, and transmembrane transport and extracellular accumulation of the heme-binding protein are realized. Through the transformation of the combination strategy, an engineering strain for efficiently producing the heme-binding protein is obtained, and the yield of the bean hemoglobin in the saccharomyces cerevisiae is remarkably improved. Under the fermentation condition of a 5L fermentation tank, the secretory expression quantity of the bean hemoglobin reaches 286.6 mg / L.

Description

Technical Field

[0001] The present invention relates to a construction technology for recombinant Saccharomyces cerevisiae strains that efficiently produce heme-binding proteins from different sources, belonging to the field of genetic engineering technology. Background Art

[0002] Heme-binding proteins (such as hemoglobin and myoglobin) have important application values in the medical, food industry, and biotechnology fields. With the rapid development of the cultured meat industry, these proteins are important coloring and flavor additives in emerging fields such as plant-based meat, and the market demand shows an exponential growth trend. Currently, the acquisition methods of heme-binding proteins mainly include extraction methods and microbial synthesis. However, traditional production methods (such as extraction from animal blood) have problems such as cumbersome extraction processes and high risks of pathogen contamination, which limit their large-scale industrial production; while the microbial heterologous expression strategy based on synthetic biology technology has become the most promising alternative due to its environmental friendliness, controllable cost, and process standardization.

[0003] Although there have been studies on the successful expression of heme-binding proteins in microorganisms such as Escherichia coli and Pichia pastoris, there are still major problems such as low-efficiency metabolic pathways, secretion problems, and poor process adaptability. Multiple rate-limiting steps in the heme synthesis process lead to insufficient heme supply, and low secretion efficiency increases the purification cost. Moreover, the existing fermentation processes cannot adapt to dynamic metabolic demands, making it difficult to achieve efficient and stable production. These problems jointly limit the industrial application of heme-binding proteins.

[0004] In terms of the selection of microbial hosts, Saccharomyces cerevisiae exhibits unique advantages: this strain not only has a mature protein expression system and an efficient recombination mechanism, but also has obtained the "Generally Recognized as Safe" (GRAS) certification from the US Food and Drug Administration (FDA), making it an ideal platform for food-grade protein production. However, S.cerevisae In the process of heme-protein complex synthesis, it faces three key bottlenecks: insufficient precursor supply and competitive consumption of metabolic flux, disordered spatial positions of heme synthase systems, and low efficiency of the secretion system. Through systematic engineering transformations such as constructing a dynamic metabolic network regulation system, designing an enzyme complex modular anchoring strategy, and optimizing the transmembrane secretion channel, the above-mentioned technical barriers have been successfully broken through. Summary of the Invention

[0005] The present invention uses S. cerevisiae CEN PK2-1C ( ΔURA3, ΔGAL80)As the starting strain, through metabolic flux redirection, dynamic regulation of modular gene clusters, and engineering of the secretion pathway, the bottlenecks of low synthesis efficiency, metabolic imbalance, and insufficient secretion ability of natural strains are broken through. A recombinant Saccharomyces cerevisiae engineering strain for efficiently producing and secreting heme-binding protein is constructed to achieve the efficient large-scale production of heme-binding protein.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A Saccharomyces cerevisiae engineering bacterium for producing heme-binding protein, and the construction method of the genetic engineering strain is as follows: (1) By integrating the α-KG transporter gene SLC25A11 with the cytoplasm IDH1 into the multi-copy site ， and regulating the expression by P TEF to obtain strain HPX01. At the same time, replace the promoter of gene Odc1p with P SUCLG1 to construct strain HPX02. Integrate ADE6 and KGDH with Hem1p into the genomic multi-copy site to construct strain HPX03. Subsequently, use the P TEF promoter to enhance the expression of ZWF1 to regenerate NADPH, and construct the ALA high-yield strain HPX04; (2) Divide the heme synthesis pathway into three modules, connect them in series through the 2A peptide chain, and regulate the co-expression of TEF1 / P ROX1 / P FRE1 promoters in stages to construct strain HPX07; LegHb (3) On the basis of HPX07, replace the signal peptide of LegHb with the α-factor leader peptide, and use P TEF to regulate and enhance the expression of SEC61 SSO1 to improve the transport efficiency, and targetedly knockout DOA4, UBP6 to inhibit degradation (HPX09), and finally obtain the engineering strain HPX10.

[0007] Preferably, the construction method of the ALA high-yield strain HPX04 is specifically as follows: A1. Construct the SLC25A11 gene overexpression cassette: 25S rDNA-up-P TEF -SLC25A11-T ADH1 -[URA3 cassette]-25S rDNA-down, and integrate it into the genomic 25S rDNA site through homologous recombination; construct the Odc1p gene overexpression cassette: POdc1p -up - [URA3 cassette] - P TEF -Odc1p - down, integrated into the genome by homologous recombination technology. This expression cassette is regulated by P TEF promoter to achieve the enrichment of α - KG in the cytoplasm. Further, construct IDH1 gene overexpression cassette: 5S rDNA - up - P TEF -IDH1 - T ADH1 -[URA3 cassette] - 5S rDNA - down. Integrated into the 5S rDNA locus by homologous recombination to construct strain HPX01 to increase the cytoplasmic α - KG concentration.

[0008] Among them, the [URA3 cassette] consists of 100bp - P TEF -URA3 - T CYC1 -100bp. After being integrated into the genome by homologous recombination, it can be negatively screened by 5 - fluoroorotic acid (5 - FOA) to achieve repeated use of multiple rounds of genetic operations in HPX strains.

[0009] A2. Replace the natural promoter of the gene SUCLG1 with P ADE6 (SEQ ID NO:5) to construct SUCLG1 expression cassette: P SUCLG1 -up - [URA3 cassette] - P ADE6 -SUCLG1 - down. Based on strain HPX01, strain HPX02 is obtained by homologous recombination technology to regulate the SUCLG1 expression level of mitochondria and further direct the carbon flux to the ALA synthesis pathway.

[0010] A3. Construct KGDH-Hem1p complex gene overexpression cassette: Ty1 - up - P TEF -KGDH - T2A - Hem1p - T ADH1 -[URA3 cassette] - Ty1 - down. Based on strain HPX02, it is integrated into the Ty1 locus by homologous recombination to construct strain HPX03 to strengthen the supply of succinyl coenzyme A and break through the rate - limiting step of Heme synthesis.

[0011] A4. Construct ZWF1 overexpression cassette: P ZWF1 -up - [URA3 cassette] - P TEF -ZWF1 - down. Based on strain HPX03, strain HPX04 is obtained by replacing the promoter through homologous recombination to promote the regeneration of NADPH.

[0012] Preferably, the construction method of the strain HPX07 is specifically as follows: B1. Based on the strain HPX04, the Heme synthesis genes and their binding proteins are concatenated into three polycistronic expression cassettes: Expression cassette 1: Ty2-up-P TEF -Hem2p-T2A-Hem3p-T2A2-Hem4p-T ADH1 -[URA3 cassette]-Ty2-down; It is integrated at the multiple-copy site of the Ty2 transposon through homologous recombination to construct the strain HPX05. Driven by the constitutive promoter P TEF and self-cleaving translation is achieved through the 2A peptide chain (T2A / T2A2) to generate independent proteins, and then the conversion of ALA to uroporphyrinogen III is catalyzed; Expression cassette 2: Delta1-up-P ROX1 -Hem12p-T2A3-Hem13p-T2A4-Hem14p-T ADH1 -[URA3cassette]-Delta1-down; It is integrated at the genomic Delta1 site through homologous recombination to construct the strain HPX06. Regulated by the hypoxia-inducible promoter P ROX1 to complete the decarboxylation and oxidation of the porphyrin ring, and the cleavage efficiency is optimized using the 2A peptide chain (T2A3 / T2A4); Expression cassette 3: Delta2-up-P FRE1 -Hem15p-T2A-LegHb-T ADH1 -[URA3 cassette]-Delta2-down; It is integrated at the multiple-copy site Delta2 through homologous recombination to construct the strain HPX07. Controlled by the Fe²⁺-inducible promoter P FRE1 to achieve the assembly and binding of Heme, and the standard T2A peptide chain is used for cleavage.

[0013] B2. Through the temporal regulation of the promoter, the imbalance of metabolic load is avoided, and at the same time, the flux distribution of intermediate products is optimized through dynamic regulation to achieve the efficient production of Heme.

[0014] Preferably, the construction method of the engineered strain HPX10 is specifically as follows: C1. Construct α-LegH an overexpression cassette: LegHb-up-α-LegHb-[URA3 cassette]-LegHb-down; Based on the strain HPX07, the native signal peptide of LegHb is replaced with the α-factor leader peptide using homologous recombination technology to obtain the strain HPX08 with improved LegHb secretion efficiency; C2. Construction SEC61, SSO1 Overexpression cassette of the gene: P SEC61 -up-[URA3 cassette]-P TEF -SEC61-down, P SSO1 -up-[URA3 cassette]-P TEF -SSO1-down; Based on the strain HPX08, it was integrated into the genome by homologous recombination technology to obtain the strain HPX09 that can enhance the vesicle transport efficiency.

[0015] C3. Construction DOA4, UBP6 Gene knockout cassette: DOA4-up-[URA3 cassette]-DOA4-down, UBP6-up-[URA3 cassette]-UBP6-down; Based on the strain HPX09, targeted gene knockout was achieved by homologous recombination technology to obtain the strain HPX10 that can inhibit extracellular proteolysis to enhance LegHb its stability.

[0016] The engineering strain HPX10 further disclosed by the present invention is applied in the production of heme proteins. Preferably, the method for preparing leghemoglobin using the engineering strain HPX10 specifically adopts a three-stage control strategy: Stage I (0 - 48 h): Glucose (20 g / L) is used as the carbon source, pH 6.0 - 6.5, dissolved oxygen 30%, rapidly accumulating α-KG to provide sufficient precursor substances for subsequent Heme synthesis. The high dissolved oxygen and sufficient carbon source at this stage promote glycolysis (EMP) and the TCA cycle, contributing to rapid cell growth and metabolism. The IDH1 overexpressed in the strain HPX04 ZWF1 and

[0017] enhance the supply of NADPH to support the cofactor requirements for heme synthesis. ROX Stage II (48 - 72 h): The dissolved oxygen is reduced to 5%. Under low oxygen conditions, the P Hem1p promoter is activated, and 0.2 mM glycine and 0.1 mM succinic acid are supplemented. Through KGDH and

[0018] catalyze the formation of ALA, enter the porphyrin synthesis pathway, and initiate the Heme metabolic flux. FRE1 Stage III (72 - 120 h): Maintain low dissolved oxygen (5%), add 0.5 mM Fe²⁺ to activate the promoter P

[0019] . Meanwhile, supplement 1 mM ALA to reduce competitive inhibition, thereby enhancing the assembly efficiency of the heme-protein complex, and finally being secreted extracellularly and accumulated under the guidance of the α-factor signal peptide.The beneficial effects of the engineered Saccharomyces cerevisiae for producing heme-binding protein, its construction method and application disclosed by the present invention are as follows: (1) Compared with other chassis microorganisms, Saccharomyces cerevisiae has a clear genetic background, mature molecular operation techniques, is easy to culture at high density, can effectively alleviate the problems of substrate and product inhibition, and improve the expression level and catalytic efficiency of rate-limiting enzymes; (2) The purpose of the present invention is to fill the gaps in the existing technology, provide an advanced strategy for strengthening the two rate-limiting steps in the upstream of heme-binding protein biosynthesis, and at the same time provide a reference for the biosynthesis of other natural products. Avoid the toxic accumulation of intermediate products in the Heme synthesis pathway, and finally achieve high-yield, high-activity and low-cost production of industrial-grade heme-binding protein. The yield of LegHb in a 5 L fermenter reaches 286.6 mg / L, which is 6 times higher than the traditional process; (3) This technology is adapted to conventional bioreactors, can be effectively extended to hosts such as Pichia pastoris, and the product can be applied to fields such as food colorants and artificial plant-based meat. At the same time, it has the advantages of high activity and environmental protection, providing an efficient, stable and sustainable solution for industrial production. Description of the Drawings

[0020] Figure 1 : Metabolic pathway of heterologous biosynthesis of heme-binding protein in Saccharomyces cerevisiae; Figure 2 : Determination results of intracellular ALA content in the starting strain, HPX01, HPX02, HPX03 and HPX04 in shake flask verification in Example 1 of the present invention; Figure 3 : Determination results of intracellular Heme content in HPX04, HPX05, HPX06 and HPX07 in shake flask verification in Example 2 of the present invention; Figure 4 : Determination results of HegHb content in the fermentation broth of HPX07, HPX08, HPX09 and HPX10 in shake flask verification in Example 3 of the present invention; Figure 5 : Expression verification of the engineered Saccharomyces cerevisiae in a 5 L fermenter. Detailed Embodiments

[0021] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0022] I. Genes involved in the present invention are as follows: Homo sapiens Mitochondrial α-KG transporter gene from SLC25A11, optimized by codons of Saccharomyces cerevisiae chassis, synthesized by Genewiz (Suzhou) Inc., and its nucleotide sequence is as shown in SEQ ID No.1; Cytosolic isocitrate dehydrogenase gene IDH1 , and its nucleotide sequence is as shown in SEQ ID No.2; Mitochondrial α-KG transporter gene Odc1p , and its nucleotide sequence is as shown in SEQ ID No.3; Succinyl-CoA synthetase gene SUCLG1 , and its nucleotide sequence is as shown in SEQ ID No.4; Cytosolic α-ketoglutarate dehydrogenase complex gene KGDH , and its nucleotide sequence is as shown in SEQ ID No.5; Aminolevulinate synthase gene Hem1p , and its nucleotide sequence is as shown in SEQ ID No.6; Glucose-6-phosphate dehydrogenase gene ZWF1 , and its nucleotide sequence is as shown in SEQ ID No.7; δ-Aminolevulinate dehydratase gene Hem2p , and its nucleotide sequence is as shown in SEQ ID NO:8; Hydroxymethylbilane synthase gene Hem3p , and its nucleotide sequence is as shown in SEQ ID NO:9; Uroporphyrinogen III synthase gene Hem4p , and its nucleotide sequence is as shown in SEQ ID NO:10; Coproporphyrinogen oxidase gene Hem12p , and its nucleotide sequence is as shown in SEQ ID NO:11; Protoporphyrinogen oxidase gene Hem13p , and its nucleotide sequence is as shown in SEQ ID NO:12; Ferrochelatase gene Hem14p , and its nucleotide sequence is as shown in SEQ ID NO:13; Heme oxygenase gene Hem15p , and its nucleotide sequence is as shown in SEQ ID NO:14; Glycine max Leghemoglobin gene from LegHb , optimized by codons of Saccharomyces cerevisiae chassis, synthesized by Genewiz (Suzhou) Inc., and its nucleotide sequence is as shown in SEQ ID NO:15; Encoding 2A peptide sequence, optimized by codons of Saccharomyces cerevisiae, and its nucleotide sequence is as shown in SEQ ID NO:16; Channel transporter subunit SEC61 , the nucleotide sequence is as shown in SEQ ID NO: 17; Synaptic fusion protease gene SSO1 , the nucleotide sequence is as shown in SEQ ID NO: 18; Gene encoding deubiquitinating enzyme DOA4 , the nucleotide sequence is as shown in SEQ ID NO: 19; Ubiquitin carboxyl-terminal hydrolase gene UBP6 , the nucleotide sequence is as shown in SEQ ID NO: 20.

[0023] II. Media and stock solutions used in the examples: YPD medium: Yeast extract 10 g / L, Tryptone 20 g / L, Glucose 20 g / L. If preparing solid medium, add 20 g / L agar additionally.

[0024] SD-URA medium: Glucose 22 g / L, YNB 6.7 g / L, Dropout Supplement 2 g / L, Leucine 100 mg / L, Tryptophan 20 mg / L, Histidine 20 mg / L. If preparing solid medium, add 20 g / L agar additionally.

[0025] 5-FoA plasmid elimination medium: Glucose 22 g / L, Yeast extract 10 g / L, Tryptone 20 g / L, Agar 20 g / L and 5-FOA 1 g / L.

[0026] Fermentation medium: Glucose 22 g / L, (NH4)2SO4 10 g / L, MgSO4 6.2 g / L, KH2PO4 8 g / L, Vitamin solution 12 mL / L, Metal ion solution 10 mL / L.

[0027] Vitamin solution (100 mL): Biotin 0.05 g / L, Calcium pantothenate 1.0 g / L, Nicotinic acid 1.0 g / L, Inositol 25 g / L, Pyridoxal hydrochloride 1.0 g / L, p-Aminobenzoic acid 0.2 g / L. Mix well, dissolve with appropriate amount of deionized water, add 1M sulfuric acid to promote dissolution, make up to 100 mL volumetric flask, and filter and sterilize with 0.22 μm filter membrane.

[0028] Metal ion solution (pH 8.0, 100 mL): 80 mL of 0.5 M EDTA. 5.75 g / L of ZnSO4·7H2O, 0.32 g / L of MnCl2·4H2O, 0.32 g / L of GuSO4, 0.47 g / L of CoCl2·6H2O, 0.48 g / L of Na2MoO4·2H2O, 2.9 g / L of CaCl2·2H2O. Mix well, dissolve with appropriate deionized water, make up the volume to 100 mL in a volumetric flask, and filter through a 0.22 μm membrane to sterilize.

[0029] 0.5 mM Fe²⁺ solution (100 mL): Weigh 2.8725 g of FeSO4·7H2O, dissolve with appropriate deionized water, make up the volume to 100 mL in a volumetric flask, and filter through a 0.22 μm membrane to sterilize.

[0030] III. Reagents used in the examples: The reagents and raw materials used in the present invention are all commercially available. Among them, the high-fidelity enzyme DNA polymerase (2×Super Pfx Master Mix) and 2×Taq Master Mix (Dye Plus) are both purchased from Nanjing Novoprotein Scientific Co., Ltd.; the DNA marker (1kb DNA ladder) and homologous recombination enzyme are purchased from Beijing TransGen Biotech Co., Ltd.; the agarose gel recovery kit is purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; the Bradford protein concentration determination kit is purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the salmon sperm DNA is purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0031] IV. Conventional technical methods used in the examples: Lithium acetate transformation method for Saccharomyces cerevisiae (1) Pick a monoclonal strain and inoculate it into a 5 mL YPD liquid test tube, and culture it overnight in a constant temperature shaker at 30 °C and 220 rpm. Transfer it to fresh YPD according to an inoculation amount of 2% (v / v) and continue to culture until the mid-logarithmic growth phase.

[0032] (2) Transfer the bacterial solution to a sterile centrifuge tube, centrifuge at 4000 rpm for 1 min, and discard the supernatant. Wash the cells with an equal volume of sterile water and 0.1 M lithium acetate solution and incubate on ice for 10 min.

[0033] (3) Add the following components in sequence to a 1.5 mL centrifuge tube: 620 μL of 50% PEG3350 solution, 90 μL of 1.0 M lithium acetate, 50 μL of salmon sperm DNA, 10 μg of exogenous plasmid DNA (or linearized DNA fragment), 100 μL of competent cells, gently mix well, and incubate in a water bath at 30 °C for 30 min.

[0034] (4) Transfer to a 42 °C water bath and heat shock for 20 - 30 min. Immediately place on ice for 2 min, centrifuge, and discard the supernatant.

[0035] (5) Resuspend the cells in 1 mL of YPD, and gently shake and recover at 30 °C for 1 - 2 h. Centrifuge to collect the cells, resuspend the cell pellet in 100 μL, and spread on SD medium. Incubate upside down at 30 °C for 2 - 3 days and observe the formation of transformants.

[0036] (6) Finally, verify whether the foreign DNA is integrated by colony PCR.

[0037] Culture method of Saccharomyces cerevisiae Plate culture: Take out the glycerol tube of the strain stored in the -80 °C refrigerator, dip a small amount of bacterial liquid with an inoculation loop and streak in three zones on the agar plate, and incubate statically in a 30 °C constant temperature incubator for 2 - 3 days.

[0038] Liquid culture: Pick a single colony from the agar plate and inoculate it into 5 mL of fresh YPD liquid medium, and incubate overnight for about 15 h in a constant temperature shaker at 30 °C and 220 rpm. Inoculate into a 250 mL Erlenmeyer flask containing 50 mL of YPD medium according to an inoculation amount of 1% (v / v), and culture at 220 rpm in a 30 °C constant temperature shaker for 120 h. In the middle and late stages of fermentation, add carbon source, 0.2 mM glycine, 0.1 mM succinic acid, and 1.0 mM hemin in sequence.

[0039] V. Detection methods ALA: Take 1 mL of the fermentation broth, centrifuge at 12,000 rpm for 5 min to remove the supernatant, resuspend the cell pellet with pH 7.4 phosphate buffer (PBS buffer), add 0.5 mm glass beads to a 2 mL microcentrifuge tube and mix, and use a cryogenic grinder to lyse the cells at 4 °C. Centrifuge the lysed mixture at 12,000 rpm for 20 min and filter through a 0.22 μm filter membrane. ALA reacts with p-dimethylaminobenzaldehyde reagent to form a red product, and measure the absorbance at 553 nm.

[0040] Heme: Determine the Heme concentration by high performance liquid chromatography. Chromatographic column: Diamonsil 5 μm C18 150 mm × 4.6 mm; column temperature: 40 °C; detector: ultraviolet detector; detection wavelength: 400 nm; mobile phase: acetonitrile and water (7:3); flow rate: 1 mL / min; injection volume: 10 μL.

[0041] LegHb: Centrifuge 1 mL of the fermentation broth at 7,000 rpm for 10 min. The supernatant is the crude extracellular enzyme solution, which is treated with a 0.22 μm filter membrane. Identify the secreted LegHb protein in the fermentation supernatant by SDS-PAGE, and determine the protein concentration using a Bradford kit.

[0042] The URA3 screening expression cassette [URA3 cassette] used in this invention is stored in our laboratory and consists of 100bp-P TEF -URA3-T CYC1 -100bp, which contains a homologous arm skeleton, P TEF promoter, URA3 screening tag, T CYC1 terminator. When changing the integration site, only 20bp in the homologous arm skeleton needs to be changed.

[0043] Example 1: Construction of high-yield ALA strain HPX04 ALA is a key precursor for Heme biosynthesis, mainly synthesized by the condensation of succinyl-CoA and glycine via the C4 pathway. The traditional synthesis pathway relies on the coordinated metabolism of mitochondrial-cytoplasmic compartmentalization, and there are problems such as insufficient precursor supply, low transmembrane transport efficiency, and competitive consumption of cofactors. In view of the above limiting factors, in this example, high-efficiency production of ALA is achieved by relieving mitochondrial dependence, optimizing precursor transmembrane transport, strengthening the expression of rate-limiting enzymes, and balancing redox homeostasis ( Figure 1 ).

[0044] Among them, SLC25A11 and Odc1p, as mitochondrial carrier proteins, are mainly involved in the exchange of substances inside and outside the mitochondria, and can transport mitochondrial α-KG to the cytoplasm, thus promoting the enrichment of α-KG in the cytoplasm and providing sufficient raw materials for ALA synthesis. At the same time, co-express IDH1 genes to catalyze the production of α-KG from isocitrate, and NADPH is generated in this process, which can further enhance the synthesis ability of α-KG. Through primers 25S rDNA-up-F / R, 25S rDNA-down-F / R, SLC25A11-F / R, P TEF (25S rDNA)-F / R, T ADH1 (25S rDNA)-F / R, URA3(25S rDNA)-F / R, amplify to obtain 25S rDNA homologous arms, SLC25A11 , promoter P TEF , terminator T ADH1 and [URA3 cassette], and obtain the SLC25A11 overexpression cassette: 25S rDNA-up-P TEF-SLC25A11-TADH1-[URA3 cassette]-25S rDNA-down, and integrated it into the 25S rDNA locus of the Saccharomyces cerevisiae genome through homologous recombination. Similarly, using primers P Odc1p -up-F / R, URA3 (Odc1p)-F / R, P TEF (Odc1p)-F / R and Odc1p-down-F / R to construct Odc1p overexpression cassette: P Odc1p -up-[URA3 cassette]-P TEF -Odc1p-down, and integrated it into the 25S rDNA locus of the Saccharomyces cerevisiae genome. In addition, using primers 5SrDNA-up-F / R, P TEF (5S rDNA)-F / R, IDH1-F / R, T ADH1 (5S rDNA)-F / R, URA3 (5S rDNA)-F / R, 5SrDNA-down-F / R to construct IDH1 overexpression cassette: 5S rDNA-up-P TEF -IDH1-T ADH1 -[URA3 cassette]-5SrDNA-down, and integrated it into the 5S rDNA locus of the Saccharomyces cerevisiae genome through homologous recombination. Through the above operations, the α-KG-enriched strain HPX01 was successfully constructed. The shake-flask fermentation results showed that compared with the ALA yield of the original strain (25.8 mg / L), the ALA yield of strain HPX01 was significantly increased to 386.5 mg / L ( Figure 2 ).

[0045] Further, regulating the expression level of mitochondrial SUCLG1 genes can optimize the cell metabolic flux, making more carbon flux direct to the ALA synthesis pathway, thereby increasing the ALA yield. The specific operation is to use primers P SUCLG1 -F / R, URA3 (SUCLG1)-F / R, P ADE6 -F / R, SUCLG1-down-F / R to construct SUCLG1 expression cassette P SUCLG1 -up-[URA3 cassette]-P ADE6 -SUCLG1-down, and then construct the metabolic flux redirection strain HPX02 through homologous recombination. The ALA yield of HPX02 was increased to 454.2 mg / L ( Figure 2 ).

[0046] Even further, there is a rate-limiting step in the Heme synthesis pathway. For KGDH-Hem1pOverexpression of the complex gene can enhance the supply of succinyl coenzyme A, breaking through the limit of natural Hem1p expression level, and then directly driving the synthesis of ALA. Through primers Ty1-up-F / R, P TEF (Ty1)-F / R, KGDH-F / R, T2A-F / R, Hem1p-F / R, T ADH1 (Ty1)-F / R, URA3(Ty1)-F / R and Ty1-down-F / R to construct KGDH-Hem1p the complex overexpression cassette: Ty1-up-P TEF -KGDH-T2A-Hem1p-T ADH1 -[URA3 cassette]-Ty1-down; Based on HPX02, it was integrated into the Ty1 locus of the Saccharomyces cerevisiae genome by homologous recombination to obtain the strain HPX03. This strain breaks through the enzyme-substrate co-efficiency bottleneck, increasing the ALA production to 646.3 mg / L ( Figure 2 ).

[0047] During the metabolic process of cells, NADPH, as an important reducing cofactor, participates in many biosynthetic reactions. ZWF1 Overexpression of the gene can promote the regeneration of NADPH, effectively maintaining the cofactor balance, thus ensuring the smooth progress of the Heme synthesis reaction. In this example, through primers P ZWF1 -F / R, URA3(ZWF1)-F / R, P TEF (ZWF1)-F / R, ZWF1-down-F / R were assembled to obtain the construction ZWF1 overexpression cassette: P ZWF1 -up-[URA3 cassette]-P TEF -ZWF1-down; Based on HPX03, it was integrated into the Saccharomyces cerevisiae genome, and the high-yield ALA strain HPX04 was successfully constructed. The NADPH regeneration ability of this strain was further improved, and the ALA production reached 712.4 mg / L ( Figure 2 ).

[0048] The above expression cassettes were transformed into Saccharomyces cerevisiae successively by the lithium acetate method, screened by SD-URA defective plates and verified by colony PCR. The successfully verified strains were inoculated into 5-FoA plasmid elimination medium and cultured at 30 °C for 24-36 h, which could effectively remove the URA3 marker plasmid in Saccharomyces cerevisiae and enter the next round of transformation. Finally, the recombinant high-yield ALA strain HPX04 was obtained: S. cerevisiae CEN PK2-1C 25S rDNA:: SLC25A11 、5S rDNA:: IDH1 、Ty1::KGDH- Hem1p 、 Odc1p ::P TEF - Odc1p 、 SUCLG1 ::P ADE6 - SUCLG1 、 ZWF1 ::P TEF - ZWF1 、Δ URA3 、Δ GAL80 。

[0049] The PCR reaction system is as follows: Component Added Volume (μL) Forword-Primer (10 μM) 2.5 Reverse-Primer (10 μM) 2.5 Template 2.0 2×Super Pfx Master Mix 25 <![CDATA[ddH2O]]> 18 PCR reaction procedure:

[0050] The primer sequences involved in Example 1 are shown in Table 1.

[0051] Primer Name Sequence Information (5’-3’) 25S rDNA-up-F GTTTGACCTCAAATCAGGTAGGAGTACC 25S rDNA-up-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGATGCTGGCCCAGTGAAATGC <![CDATA[P TEF (25S rDNA)-F]]> GCATTTCACTGGGCCAGCATCCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC <![CDATA[P TEF (25S rDNA)-R]]> GATGCGGTTGCTGCCATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC SLC25A11-F GCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGATGGCAGCAACCGCATC SLC25A11-R CACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTCCGGATAGAAATAATCTTTTATAAGCCTTGTTC <![CDATA[T ADH1 (25S rDNA)-F]]> GAACAAGGCTTATAAAAGATTATTTCTATCCGGAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG <![CDATA[T ADH1 (25S rDNA)-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATAACGGAGCGACCTCATGCTATACCTGAG URA3 (25S rDNA)-F CTCAGGTATAGCATGAGGTCGCTCCGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3 (25S rDNA)-R GCTTACCGAATTCTGCTTCGGTATTCTTGTTGAGGGTCACTCAATTGAACTTC 25S rDNA-down-F GAAGTTCAATTGAGTGACCCTCAACAAGAATACCGAAGCAGAATTCGGTAAGC 25S rDNA-down-R ACAAATCAGACAACAAAGGCTTAATCTCAG <![CDATA[P Odc1p -up-F]]> GCGTTGAAATGAAGGAAATAATGAAAGTCTAG <![CDATA[P Odc1p -up-R]]> CTGTAATCTGCCTTCTTATTCAAAGATAACGACAAGAACAAGATCAAACCCTCACTAATCAAC URA3 (Odc1p)-F GTTGATTAGTGAGGGTTTGATCTTGTTCTTGTCGTTATCTTTGAATAAGAAGGCAGATTACAG URA3 (Odc1p)-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGTCTTGTTGAGGGTCACTCAATTGAACTTC <![CDATA[P TEF (Odc1p)-F]]> GAAGTTCAATTGAGTGACCCTCAACAAGACATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC <![CDATA[P TEF (Odc1p)-R]]> GAACGGCAAAGGTCTATTATCTATAGATGTCATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC Odc1p-down-F GCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGATGACATCTATAGATAATAGACCTTTGCCGTTC Odc1p-down-R CTTCACTAGTTCAAAAGGCGCGAC 5S rDNA-up-F AAGCTTCTTCTCTCAGGAGGCC 5S rDNA-up-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGAAAATATCTTTAGTGCATTGGTACAGTGGTAG <![CDATA[P TEF (5S rDNA)-F]]> CTACCACTGTACCAATGCACTAAAGATATTTTCCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTT C <![CDATA[P TEF (5S rDNA)-R]]> CTAAAGTTCTCTTAGCAATTGTTCTGTTAAGCATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC IDH1-F GCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGATGCTTAACAGAACAATTGCTAAGAGAACTTTAG IDH1-R CACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCCATGGTAGATAATTTGTTGATGATTTCATTCGTGAAG <![CDATA[T ADH1 (5S rDNA)-F]]> CTTCACGAATGAAATCATCAACAAATTATCTACCATGGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG <![CDATA[T ADH1 (5S rDNA)-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATAACGGAGCGACCTCATGCTATACCTGAG URA3 (5S rDNA)-F CTCAGGTATAGCATGAGGTCGCTCCGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3 (5S rDNA)-R CAAGGGATTGAACGGCATATTTTGAACTCTTGTTGAGGGTCACTCAATTGAACTTC 5S rDNA-down-F GAAGTTCAATTGAGTGACCCTCAACAAGAGTTCAAAATATGCCGTTCAATCCCTTG 5S rDNA-down-R AAGCTTAACCTTCCAAAAAGTGTTGTCC <![CDATA[P SUCLG1 -up-F]]> GCGTTTCTCGAGACTGATATCCAGATG <![CDATA[P SUCLG1 -up-R]]> CTGTAATCTGCCTTCTTATTCAAAGATAACGTTATCAAACTGAAGAATAAATGGTCTGC URA3 (SUCLG1)-F GCAGACCATTTATTCTTCAGTTTGATAACGTTATCTTTGAATAAGAAGGCAGATTACAG URA3 (SUCLG1)-R CAACCGAGTCTCGATACGTTCAGTCTTGTTGAGGGTCACTCAATTGAACTTC <![CDATA[P ADE6 -F]]> GAAGTTCAATTGAGTGACCCTCAACAAGACTGAACGTATCGAGACTCGGTTG <![CDATA[P ADE6 -R]]> GAGAAGCTTTTGAAACGGTAGATCTTAACATTGAACTTGACTTCTTTTGTTATGGACCTGG SUCLG1-down-F CCAGGTCCATAACAAAAGAAGTCAAGTTCAATGTTAAGATCTACCGTTTCAAAAGCTTCTC SUCLG1-down-R CTGGCAGTGGCTCCAGTC Ty1-up-F TCCGCGCTGAGGGTTTAATGG Ty1-up-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGATATGTTTATATTCATTGATCCTATTACATTATCAATCCTTGC <![CDATA[P TEF (Ty1)-F]]> GCAAGGATTGATAATGTAATAGGATCAATGAATATAAACATATCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC <![CDATA[P TEF (Ty1)-R]]> GCGAAGACACGAACCTTAGCATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC KGDH-F GCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGATGCTAAGGTTCGTGTCTTCGC KGDH-R GTCAGAAGAGAACCACGACCTTCACAATGAATAGCTTTATCATAGGCAGCC T2A-F GGCTGCCTATGATAAAGCTATTCATTGTGAAGGTCGTGGTTCTCTTCTGAC T2A-R GTTTTGGCTTGCAGATTTCAACATTGGACCTGGGTTTTCTTCAACATC Hem1p-F GATGTTGAAGAAAACCCAGGTCCAATGTTGAAATCTGCAAGCCAAAAC Hem1p-R CACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTGTTAAGCTTCTCTTGGGAATTGTTGGG <![CDATA[T ADH1 (Ty1)-F]]> CCCAACAATTCCCAAGAGAAGCTTAACAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG <![CDATA[T ADH1 (Ty1)-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATAACGGAGCGACCTCATGCTATACCTG URA3 (Ty1)-F CAGGTATAGCATGAGGTCGCTCCGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3 (Ty1)-R CATACTAATATTACGATTATTCCTCATTCCGTTTTTCTTGTTGAGGGTCACTCAATTGAACTTC Ty1-down-F GAAGTTCAATTGAGTGACCCTCAACAAGAAAAACGGAATGAGGAATAATCGTAATATTAGTATG Ty1-down-R AGTATAGGAACTTCACTTCAGGTCTGAGT <![CDATA[P ZWF1 -up-F]]> GCCGCGGTCAGTGACATTTTG <![CDATA[P ZWF1 -up-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATA ACGGGGCCGAGCGCCG URA3 (ZWF1)-F CGGCGCTCGGCCCCGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3 (ZWF1)-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGTCTTGTTGAGGGTCACTCAATTGAACTTC <![CDATA[P TEF (ZWFI)-F]]> GAAGTTCAATTGAGTGACCCTCAACAAGACATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC <![CDATA[P TEF (ZWF1)-R]]> GACGGGGCCTTCACTCATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC ZWF1-down-F GCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGATGAGTGAAGGCCCCGTC ZWF1-down-R TCTGCGTTCTTAATTCGTCGAAGC Example 2: Construction of modular heme-producing strain HPX07 Currently, in Saccharomyces cerevisiae, the synthesis pathway from ALA to Heme faces many challenges. This pathway involves multiple enzymatic steps catalyzed by enzymes encoded by different genes, but there is a lack of coordination in the expression time and level of these genes, forming a metabolic bottleneck. In addition, the insufficient accumulation of intermediate products will interfere with the metabolic network balance and affect cell growth and product synthesis efficiency. Therefore, in this example, strategies such as constructing three polycistronic expression cassettes, promoter temporal regulation, and dynamic expression are adopted to solve these problems ( Figure 1 ).

[0052] Among them, expression cassette 1 is driven by the constitutive promoter P TEF to express, and T2A ensures efficient cleavage of independent proteins. These genes are involved in the early steps of Heme synthesis, converting ALA into uroporphyrinogen III. Expression cassette 1: Ty2-up-P Hem2p, Hem3p, Hem4p is constructed through primers Ty2-up-F / R, P TEF (Ty2)-F / R, Hem2p-F / R, T2A (Ty2)-F / R, Hem3p-F / R, T2A2 (Ty2)-F / R, Hem4p-F / R, T ADH1 (Ty2)-F / R, URA3 (Ty2)-F / R, and Ty2-down-F / R: Ty2-up-P TEF -Hem2p-T2A-Hem3p-T2A2-Hem4p-T ADH1-[URA3 cassette]-Ty2-down; Based on strain HPX04, it was integrated into the Ty2 transposon site through homologous recombination technology to construct strain HPX05, and the Heme production increased from 11.8 mg / L (HPX04) to 25.6 mg / L ( Figure 3 ).

[0053] Expression cassette 2 is regulated by the hypoxia-inducible promoter P ROX1 regulation Hem12p, Hem13p, Hem14p expression, a yeast codon-optimized T2A variant with a cleavage efficiency > 92%. These genes are involved in the intermediate steps of Heme synthesis and complete the decarboxylation and oxidation of the porphyrin ring. By using primers Delta1-up-F / R, P ROX1 -F / R, Hem12p-F / R, T2A3(Delta1)-F / R, Hem13p-F / R, T2A4(Delta1)-F / R, Hem14p-F / R, T ADH1 (Delta1)-F / R, URA3(Delta1)-F / R and Delta1-down-F / R to construct expression cassette 2: Delta1-up-P ROX1 -Hem12p-T2A3-Hem13p-T2A4-Hem14p-T ADH1 -[URA3 cassette]-Delta1-down; Based on strain HPX05, it was integrated into the Delta1 site through homologous recombination technology to construct strain HPX06. The integration of expression cassette 2 further increased the production of uroporphyrinogen III, optimized the flux of intermediate products in the Heme synthesis pathway, and increased the Heme production to 76.9 mg / L ( Figure 3 )

[0054] Expression cassette 3 is controlled by the Fe 2+ -inducible promoter P FRE1 control Hem15p and LegHb expression, and through standard T2A peptide cleavage, these genes are involved in the later steps of Heme synthesis to achieve the assembly of Heme and its binding to hemoglobin. By using primers Delta2-up-F / R, P FRE1 -F / R, Hem15p-F / R, T2A(Delta2)-F / R, LegHb-F / R, T ADH1 (Delta2)-F / R, URA3(Delta2)-F / R and Delta2-down-F / R to construct expression cassette 3: Delta2-up-P FRE1 -Hem15p-T2A-LegHb-T ADH1-[URA3 cassette]-Delta2-down; Based on strain HPX06, it was integrated into the multi-copy site Delta2 by homologous recombination to construct strain HPX07. In particular, the Heme production of HPX07 was significantly increased, and the Heme production increased to 116.8 mg / L ( Figure 3 ).

[0055] Furthermore, by regulating the promoter timing to avoid metabolic load imbalance, the constitutive promoter of expression cassette 1 ensures the continuous progress of early steps, the hypoxia-inducible promoter of expression cassette 2 is activated under hypoxia to optimize the flux distribution of intermediate products, and the iron ion-inducible promoter of expression cassette 3 is activated by Fe 2+ to ensure the efficient progress of late steps. Finally, dynamic regulation was used to optimize the flux distribution of intermediate products, prevent their excessive accumulation, and improve the overall efficiency of Heme synthesis. Through these strategies, the dynamic expression strains HPX05, HPX06, and HPX07 were successfully constructed, effectively solving the problem of ALA to Heme synthesis and increasing Heme production.

[0056] Strain HPX07: S. cerevisiae CEN PK2-1C 25S rDNA:: SLC25A11 、5S rDNA:: IDH1 、Ty1:: KGDH-Hem1p、 Ty2:: Hem2p-Hem3p-Hem4p、 Delta1:: Hem12p-Hem13p-Hem14p、 Delta2:: Hem15p-LegHb, Odc1p ::P TEF - Odc1p 、 SUCLG1 ::P ADE6 - SUCLG1 、 ZWF1 ::P TEF - ZWF1, ΔURA3, Δ GAL80 。

[0057] The primer sequences involved in this Example 2 are shown in Table 2.

[0058] Primer Name Sequence Information (5'-3') Ty2-up-F GTGTCCGCGCTGAGGG Ty2-up-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTTAATATTCATTGATCCTATTACATTATCAATCCTTGCG <![CDATA[P TEF (Ty2)-F]]> CGCAAGGATTGATAATGTAATAGGATCAATGAATATTAACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC <![CDATA[P TEF (Ty2)-R]]> CTGTTTCCAAAAATTCAGCTGTATGCATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC Hem2p-F GCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGATGCATACAGCTGAATTTTTGGAAACAG Hem2p-R CAAGTCAGAAGAGAACCACGACCTTCGTTTTCTTCATCTAACCAGTCTAGGAACTCAG T2A(Ty2)-F CTGAGTTCCTAGACTGGTTAGATGAAGAAAACGAAGGTCGTGGTTCTCTTCTGACTTG T2A(Ty2)-R CAATATGTAGAGTTTCAGGGCCCATTGGACCTGGGTTTTCTTCAACATCAC Hem3p-F GTGATGTTGAAGAAAACCCAGGTCCAATGGGCCCTGAAACTCTACATATTG Hem3p-R CAAGTAAGTAGGGATCCACGACCTTCTCATTTGATTCTGTCTAAATTAATTTCATCCAGAATTTTCTTTG T2A2(Ty2)-F CAAAGAAAATTCTGGATGAAATTAATTTAGACAGAATCAAATGAGAAGGTCGTGGATCCCTACTTACTTG T2A2(Ty2)-R GCAGTAATACTCTGACTTTTTTACGACTAGACATCGGACCAGGGTTTTCCTCTAC Hem4p-F GTAGAGGAAAACCCTGGTCCGATGTCTAGTCGTAAAAAAGTCAGAGTATTACTGC Hem4p-R CACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTTTATGGCGCTGGTATAATTCAATAGCATC <![CDATA[T ADH1 (Ty2)-F]]> GATGCTATTGAATTATACCAGCGCCATAAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG <![CDATA[T ADH1 (Ty2)-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATAACGGAGCGACCTCATGCTATACCTGAG URA3 (Ty2)-F CTCAGGTATAGCATGAGGTCGCTCCGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3 (Ty2)-R GAATCTGCAATTCTACACAATTCTATAAATATTATTATCATCATTTTATATTCTTGTTGAGGGTCACTCAATTGAACTTC Ty2-down-F GAAGTTCAATTGAGTGACCCTCAACAAGAATATAAAATGATGATAATAATATTTATAGAATTGTGTAGAATTGCAGATTC Ty2-down-R GTATAGGAACTTCACTTCAGGTCTGAGTG Delta1-up-F GGCTGGCAACTAATAGGGACACTAC Delta1-up-R GGAATCTTAGGTGTAGAGGATTTAGGATTCATGATTATTCCTCATTCCGTTTTATATGTTTCATTATCC <![CDATA[P ROX1 -F]]> GGATAATGAAACATATAAAACGGAATGAGGAATAATCATGAATCCTAAATCCTCTACACCTAAGATTCC <![CDATA[P ROX1 -R]]> GTTTTTTGGAGCTGGAAAGTTACCCATTTTCGGAGAAACTAGGCTAGTTTTAGC Hem12p-F GCTAAAACTAGCCTAGTTTCTCCGAAAATGGGTAACTTTCCAGCTCCAAAAAAC Hem12p-R CAAGTAAGCAAAGAACCTCTACCCTCCTTCGAACCAATTCTGTGGCACTC T2A3 (Delta1)-F GAGTGCCACAGAATTGGTTCGAAGGAGGGTAGAGGTTCTTTGCTTACTTG T2A3 (Delta1)-R GGATCTTGAGGGGCAGGCATTGGACCTGGGTTTTCCTCAAC Hem13p-F GTTGAGGAAAACCCAGGTCCAATGCCTGCCCCTCAAGATCC Hem13p-R CAAAAGGGATCCACGTCCTTCTTTAACCCACTCTCTTGGTTTGGTG T2A4 (Delta1)-F CACCAAACCAAGAGAGTGGGTTAAAGAAGGACGTGGATCCCTTTTG T2A4 (Delta1)-R CTCTCGGTTTTAGCTTTGTTAATGGTAATAACATAGGACCTGGATTCTCTTCGACATC Hem14p-F GATGTCGAAGAGAATCCAGGTCCTATGTTATTACCATTAACAAAGCTAAAACCGAGAG Hem14p-R CACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTTTGCTTAGCTGTAAGGCGTCG <![CDATA[T ADH1 (Delta1)-F]]> CGACGCCTTACAGCTAAGCAAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG <![CDATA[T ADH1 (Delta1)-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATAACGGAGCGACCTCATGCTATACCTGAG URA3 (Delta1)-F CTCAGGTATAGCATGAGGTCGCTCCGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3 (Delta1)-R GGAATCCTCAAAATGGAATCTTTATCTCTATATACTAATATTACTCTTGTTGAGGGTCACTCAATTGAACTTC Delta1-down-F GAAGTTCAATTGAGTGACCCTCAACAAGAGTAATATTAGTATATAGAGATAAAGATTCCATTTTGAGGAT TCC Delta1-down-R AGGCTATAATATCAGGTATACAGAATATACTAGAAGTTC Delta2-up-F TGTTGGAATAAAAATCAACTATCATCTACAACTAGTATTTAC Delta2-up-R CTGCATGAAGGCACGCTCTAATATCCACTAATTTAGATGACTATTTCTCATCATTTGC <![CDATA[P FRE1 -F]]> GCAAATGATGAGAAATAGTCATCTAAATTAGTGGATATTAGAGCGTGCCTTCATGCAG <![CDATA[P FRE1 -R]]> GTGTACGGATTGTTCTGGAAAGCATTATTGATCCGGCTCGTTCTTCCG Hem15p-F CGGAAGAACGAGCCGGATCAATAATGCTTTCCAGAACAATCCGTACAC Hem15p-R GTCAGAAGAGAACCACGACCTTCTCAAGTAGATTCGTGATTGCCAAATACC T2A(Delta2)-F GGTATTTGGCAATCACGAATCTACTTGAGAAGGTCGTGGTTCTCTTCTGAC T2A(Delta2)-R GTCTTGTTTTTCGGTAAATGCAACCATTGGACCTGGGTTTTCTTCAACATC LegHb-F GATGTTGAAGAAAACCCAGGTCCAATGGTTGCATTTACCGAAAAACAAGAC LegHb-R CACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCAGCTTTCTTTATAGCAGCTGCTAATTCG <![CDATA[T ADH1 (Delta2)-F]]> CGAATTAGCAGCTGCTATAAAGAAAGCTGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG <![CDATA[T ADH1 (Delta2)-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATAACGGAGCGACCTCATGCTATACCTG URA3(Delta2)-F CAGGTATAGCATGAGGTCGCTCCGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3(Delta2)-R CATTATCAATCCTTGCGTTTCAGCTTCTTGTTGAGGGTCACTCAATTGAACTTC Delta2-down-F GAAGTTCAATTGAGTGACCCTCAACAAGAAGCTGAAACGCAAGGATTGATAATG Delta2-down-R AAAAGGGAATCTGCAATTCTACACAATTCT Example 3: Construction of Secretion Engineering Bacterium HPX10 In Saccharomyces cerevisiae, the secretion of leghemoglobin LegHb depends on the classical eukaryotic secretion pathway, which is guided by a signal peptide into the endoplasmic reticulum for folding and modification, processed by vesicle transport to the Golgi apparatus, and finally secreted extracellularly through membrane fusion. However, there are many challenges in this process, such as the low efficiency of natural signal peptides, the low efficiency of vesicle transport, and the easy degradation of extracellular proteins. To address the above problems, in this example, by replacing LegHb the natural signal peptide, the transmembrane transport and vesicle transport efficiency of proteins were enhanced, and the degradation of extracellular proteins was inhibited (Figure 1 ). The secretion efficiency and stability of LegHb were successfully improved, ultimately increasing the production of hemopexin.

[0059] Among them, hemopexin is responsible for the transport and storage of Heme. To improve the secretion efficiency of LegHb protein, its native signal peptide was replaced with the α-factor leader peptide. Using primers LegHb-up-F / R, α-MF-F / R, LegHb-F / R, URA3(α-MF)-F / R, and LegHb-down-F / R to construct α-LegHb Expression cassette: LegHb-up-α-LegHb-[URA3 cassette]-LegHb-down; Based on the strain HPX07, the native signal peptide of LegHb protein was replaced by homologous recombination technology to construct the strain HPX08. The shake-flask fermentation results showed that the secretion efficiency of LegHb was improved, and the extracellular protein concentration increased from 10.7 mg / L of HPX07 to 28.5 mg / L ( Figure 4 ).

[0060] Furthermore, to enhance the efficiency of protein transmembrane transport and vesicular transport, overexpress the genes SEC61 and SSO1 . SEC61 Encodes a subunit of the translocase complex on the endoplasmic reticulum membrane and is involved in protein transmembrane transport; SSO1 Encodes a vesicle fusion protein between the endoplasmic reticulum and the Golgi apparatus and is involved in vesicular transport. Using primers P SEC61 -F / R, URA3(SEC61)-F / R, P TEF (SEC61)-F / R, and SEC61-down-F / R to construct an overexpression cassette: P SEC61 -up-[URA3 cassette]-P TEF -SEC61-down; Using primers P SSO1 -F / R, URA3(SSO1)-F / R, P TEF (SSO1)-F / R, and SSO1-down-F / R to construct an overexpression cassette: P SSO1 -up-[URA3 cassette]-P TEF -SSO1-down. Based on the strain HPX08, the above two overexpression cassettes were integrated into the genome by homologous recombination technology to obtain the strain HPX09. The vesicular transport efficiency of HPX09 was significantly enhanced, and the shake-flask fermentation yield of LegHb was further increased to 42.2 mg / L ( Figure 4 ).

[0061] Even further, to inhibit extracellular protein degradation and improve protein stability, knockout the geneDOA4 and UBP6 to reduce the degradation of LegHb protein and enhance the stability of LegHb. The DOA4 protein is involved in protein ubiquitination and degradation. The UBP6 protein is involved in protein deubiquitination. Knockout cassette DOA4-up-[URA3 cassette]-DOA4-down was constructed using primers DOA4-up-F / R, URA3(DOA4)-F / R, and DOA4-down-F / R; knockout cassette UBP6-up-[URA3 cassette]-UBP6-down was constructed using primers UBP6-up-F / R, URA3(UBP6)-F / R, and UBP6-down-F / R. Based on strain HPX09, targeted gene knockout was achieved through homologous recombination to construct strain HPX10. Finally, the shake flask fermentation yield of LegHb was increased to 62.3 mg / L ( Figure 4 ), and these improvement measures significantly enhanced the secretion efficiency and stability of LegHb, thereby increasing the yield of heme-binding protein.

[0062] Finally, the engineered yeast strain HPX10 secreting leghemoglobin was obtained: S. cerevisiae CEN PK2-1C 25SrDNA:: SLC25A11、 5S rDNA:: IDH1、 Ty1:: KGDH-Hem1p、 Ty2:: Hem2p-Hem3p-Hem4p、 Delta1::He m12p-Hem13p-Hem14p Delta2:: Hem15p-α-LegHb、Odc1p :: P TEF - Odc1p 、 SUCLG1 ::P ADE6 - SUCLG1 、 ZWF1 ::P TEF - ZWF1 、 SEC61 :: P TEF -SEC61 、 SSO1 :: P TEF -SSO1 、 ΔURA3、Δ GAL80、ΔDOA4、ΔUBP6 。

[0063] The primer sequences involved in Example 3 are shown in Table 3 Primer Name Sequence information (5'-3') LegHb-up-F CGGGGTACCATGGTTGCATTTACCGAAAAACAAGAC LegHb-up-R CGAATAAAACTGCAGTAAAAATTGAAGGAAATCTCATGGCCTTCAGTTGTCCGGC α-MF-F GCCGGACAACTGAAGGCCATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCG α-MF-R CGTCTTGTTTTTCGGTAAATGCAACCATAGCTTCAGCCTCTCTTTTCTCGA LegHb-F TCGAGAAAAGAGAGGCTGAAGCTATGGTTGCATTTACCGAAAAACAAGACG LegHb-R GACTGTAATCTGCCTTCTTATTCAAAGATAACGTTAAGCTTTCTTTATAGCAGCTGCTAATTC URA3(α-MF)-F GAATTAGCAGCTGCTATAAAGAAAGCTTAACGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3(α-MF)-R CGGCCACAACAGTGCCAGATCTTGTTGAGGGTCACTCAATTGAACTTC LegHb-down-F GAAGTTCAATTGAGTGACCCTCAACAAGATCTGGCACTGTTGTGGCCG LegHb-down-R GGAGCTCAGCTTTCTTTATAGCAGCTGCTAATTCG <![CDATA[P SEC61 -up-F]]> CTAACCGCCGATTAACAATATCATACGC <![CDATA[P SEC61 -up-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATAACGCTACTAAGAATAATAAAATACCCCGGAGGTG URA3(SEC61)-F CACCTCCGGGGTATTATTATTATTCTTAGTAGCGTTATCTTTGAATAAGAGGCAGATTACAGTC URA3(SEC61)-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGTCTTGTTGAGGTCACTCAATTGAACTTC <![CDATA[P TEF (SEC61)-F]]> GAAGTTCAATTGAGTGACCCTCAACAAGACATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC <![CDATA[P TEF (SEC61)-R]]> CAAGTCTAGAACACGGTTGGAGGACATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC SEC61-down-F GCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGATGTCCTCCAACCGTGTTCTAGACTTG SEC61-down-R GGAGCTCCCCTGCAAAAATTGGAAAATCATAGATGAAGT <![CDATA[P SSO1 -up-F]]> GACAAAAGACGATATGCAGGCTGTG <![CDATA[P SSO1 -up-R]]> GACTGTAATCTGCCTTCTTATTCAAAGATAACGTTTGATTTGTTTCTATTTTTAATTGCCTTTTTTAATTGTAAAAGGG URA3(SSO1)-F CCCTTTTACAATTAAAAAAGGCAATTAAAAATAGAAACAAATCAAACGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3(SSO1)-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGTCTTGTTGAGGGTCACTCAATTGAACTTC <![CDATA[P TEF (SSO1)-F]]> GAAGTTCAATTGAGTGACCCTCAACAAGACATAGCTTCAAAATGTTTCTCTCCTTTTTTACTCTTC <![CDATA[P TEF (SSO1)-R]]> GTTTCCAACTGGTACGGATTATTATAACTCATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC SSO1-down-F GCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGATGAGTTATAATAATCCGTACCAGTTGGAAAC SSO1-down-R GGAGCTCAGTCCGTGGCTTGTGCGA DOA4-up-F CGGGGTACCATGGAGCAGAATATTATTAGTACCATAAGGGATG DOA4-up-R GACTGTAATCTGCCTTCTTATTCAAAGATAACGTGCACCCTTATTTTTGCACGATATAATATTTTTG URA3(DOA4)-F CAAAAATATTATATCGTGCAAAAATAAGGGTGCACGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3(DOA4)-R CCTGGAGGAAAAACCCCGTCTCTTGTTGAGGGTCACTCAATTGAACTTC DOA4-down-F GAAGTTCAATTGAGTGACCCTCAACAAGAGACGGGGTTTTTCCTCCAGG DOA4-down-R GGAGCTCAACACCGTAGACGCGGTGATAAA UBP6-up-F CGGGGTACCATGAGCGGAGAAACGTTTGAGTTCAATATTAG UBP6-up-R GACTGTAATCTGCCTTCTTATTCAAAGATAACGGTTAGCATCTGGAGTCCCCAATAGC URA3(UBP6)-F GCTATTGGGGACTCCAGATGCTAACCGTTATCTTTGAATAAGAAGGCAGATTACAGTC URA3(UBP6)-R GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGTCTTGTTGAGGTCACTCAATTGAACTTC UBP6-down-F GAAGTTCAATTGAGTGACCCTCAACAAGACATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC UBP6-down-R GGAGCTCCAGACCAAATCCTTTATACATTAAGATCAGTGC Example 4: Optimization of the staged fermentation process Through the transformation of the combination strategy in the above embodiments, an engineered strain capable of efficiently producing heme-binding protein was successfully obtained. High-density fermentation was carried out using a fed-batch strategy in a 5 L fermenter, and the process was divided into three stages: Stage I (0 - 48 hours): Using 20 g / L glucose as the carbon source, controlling the pH at 6.0 - 6.5 and the dissolved oxygen at 30%, continuously adding glucose (maintaining the residual sugar > 5 g / L), and automatically adding ammonia water. Promote the EMP and TCA cycles through high dissolved oxygen and sufficient carbon source, and the HPX10 strain accumulates biomass and α-KG, and the OD 600 reaches 115.3. At the same time, in the strain HPX10 IDH1 and ZWF1 , supply NADPH regeneration, and the constitutive promoter P TEF drives Hem2p, Hem3p, Hem4p ; Stage II (48 - 72 hours): Add 0.2 mM glycine and 0.1 mM succinic acid. Through Hem1p and KGDH synergistically catalyze the formation of ALA, and the accumulation of the Heme precursor increases significantly. The dissolved oxygen drops to 5%, and the low oxygen condition activates P ROX , Hem12p, Hem13p, Hem14p expression is up-regulated. Convert ALA into porphyrin ring intermediates. The cells are slightly autolyzed, the biomass decreases, and the OD 600 stabilizes at 108 ( Figure 5 ); Stage III (72 - 120 hours): Maintain the dissolved oxygen at 5%, add 0.5 mM Fe²⁺ and 1 mM ALA, activate the P FRE1 promoter, Hem15p and LegHb expression is up-regulated, and strengthen the assembly of Heme and leghemoglobin LegHb. The α-factor signal peptide guides secretion. Finally, after 120 hours of fermentation, the secreted yield of leghemoglobin LegHb reaches 286.6 mg / L ( Figure 5 ), and the secretion efficiency is 3.5 times higher than that of the parental strain, and the extracellular half-life is extended to 72 hours.

[0064] The present invention adopts a modular dynamic regulation technology, combines iron ion induction and fed-batch fermentation strategy, and significantly improves the assembly efficiency and extracellular stability of the heme-protein complex. Using Saccharomyces cerevisiae as the chassis host, through metabolic engineering and secretion system optimization, the efficient production and secretion of heme-binding protein are realized.

[0065] In summary, the yeast recombinant strain of the present invention has high stability, can directly biosynthesize leghemoglobin, its yield is higher than that of the reported engineered strains, the production cost is lower, it is green and environmentally friendly, and has social benefits, and can be applied industrially.

[0066] The embodiments of the present invention described above are not intended to limit the present invention, and any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant Saccharomyces cerevisiae Saccharomyces cerevisiae CEN.PK2-1C engineering strain, characterized in that Efficient production and secretion of heme-binding proteins are achieved through the following modifications: (1) Through metabolic flux redirection, α-ketoglutaric acid (α-KG) is enriched in the cytoplasm, and the rate of oxidative decarboxylation reaction to generate succinyl coenzyme A is accelerated, and then it combines with glycine to generate 5-aminolevulinic acid (ALA); (2) Modularly integrate the heme synthesis gene cluster and its binding proteins into the Saccharomyces cerevisiae genome and dynamically regulate the gene expression level; (3) Optimize the secretion expression system to achieve efficient transmembrane transport and extracellular stable accumulation of heme-binding proteins.

2. The recombinant Saccharomyces cerevisiae according to claim 1 Saccharomyces cerevisiae The engineered strain CEN.PK2-1C, characterized in that The construction method described in step (1) is as follows: A1. Integrate the mitochondrial α-KG transporter gene at the rDNA multi-copy site SLC25A11 (SEQ ID NO:1) and the gene encoding cytoplasmic isocitrate dehydrogenase IDH1 (SEQ ID NO:2), and overexpress TEF under the control of the P Odc1p (SEQ ID NO:3) gene to construct the α-KG enriched strain HPX01; A2. On the basis of the strain HPX01, replace the mitochondrial succinyl-CoA synthetase gene SUCLG1 The natural promoter of (SEQ ID NO: 4) is P ADE6 , and construct a strain with redirected metabolic flux, strain HPX02; Based on the strain HPX02, the gene encoding the cytoplasmic α-KG dehydrogenase complex is integrated at the Ty transposon site KGDH (SEQ ID NO:5) and the 5-ALA synthase gene Hem1p (SEQ ID NO:6) to construct the ALA-accumulating strain HPX03; A4. On the basis of the strain HPX03, use the P TEF promoter to overexpress the glucose-6-phosphate dehydrogenase gene ZWF1 (SEQ ID NO:7) to accelerate NADPH regeneration and meet the requirements of the heme synthesis pathway, and construct the high-ALA-producing strain HPX04.

3. The recombinant Saccharomyces cerevisiae according to claim 1 Saccharomyces cerevisiae The engineered strain CEN.PK2-1C, characterized in that The steps described in step (2) are as follows: B1. Based on the strain HPX04, divide the heme synthesis gene cluster and its binding proteins into three functional modules and integrate them into the Ty transposon sequence and the Delta site; Module 1: Hem2p - Hem3p - Hem4p (SEQ ID NO:8-10), catalyzing the conversion of ALA to uroporphyrinogen III; Module 2: Hem12p - Hem13p - Hem14p (SEQ ID NO: 11-13)), completing the decarboxylation and oxidation of the porphyrin ring; Module 3: Hem15p - LegHb (SEQ ID NO:14-15), which realizes the assembly and binding of heme; B2. Each module within the gene cluster is concatenated by a 2A peptide chain (SEQ ID NO: 16) and is dynamically regulated by promoters P TEF1 , P ROX1 , and P FRE1 respectively to achieve stage expression, and strains HPX05, HPX06, and HPX07 are constructed.

4. The recombinant Saccharomyces cerevisiae according to claim 1 Saccharomyces cerevisiae The engineered strain CEN.PK2-1C, characterized in that The operation of optimizing the secretion system described in step (3) is as follows: C1. Based on the strain HPX07, replace the natural signal peptide of the heme-binding protein LegHb with the α-factor leader peptide to construct the strain HPX08; C2. On the basis of the strain HPX08, the promoter expresses and regulates the endoplasmic reticulum-Golgi transport protein genes TEF (SEQ ID NO:17) and SEC61 (SEQ ID NO:18), and the strain HPX09 is constructed; SSO1 ​ Based on the strain HPX09, genes related to exosome degradation were knocked out DOA4 (SEQ ID NO:19) and UBP6 (SEQ ID NO:20) to construct the strain HPX10.

5. The recombinant Saccharomyces cerevisiae according to claim 2-4 Saccharomyces cerevisiae The engineered strain CEN.PK2-1C, characterized in that Any of the said genes can be replaced with homologous genes; the promoters include, but are not limited to: P ADE6 , P TEF1 , P ROX1 , P FRE1 ; the module connection methods include, but are not limited to, 2A peptide chains; the integration sites include, but are not limited to: rDNA region, Delta site, Ty transposon site, HO site.

6. A method for producing a secreted heme-binding protein, characterized in that The recombinant Saccharomyces cerevisiae according to any one of claims 1-4 Saccharomyces cerevisiae The CEN.PK2-1C engineering strain, and its fermentation process includes: Phase I (0 - 48 h): The carbon source is glucose (20 g / L), pH 6.0 - 6.5, dissolved oxygen 30%; Phase II (48 - 72 h): Reduce the dissolved oxygen to 5%, and supplement 0.2 mM glycine and 0.1 mM succinic acid; Phase III (72 - 120 h): Maintain the dissolved oxygen at 5%, and supplement 0.5 mM Fe²⁺ and 1 mM ALA.

7. A yeast strain secreting hemopexin, characterized in that, Based on any one of claims 1 - 6, heterologously express leghemoglobin LegHb; the heme-binding protein includes but is not limited to LegHb.

8. The recombinant Saccharomyces cerevisiae according to any one of claims 1-7 Saccharomyces cerevisiae The engineered strain CEN.PK2-1C, characterized in that The strain or method can be extended to other microbial hosts, including but not limited to: Pichia pastoris ( Pichia pastoris ), Yarrowia lipolytica ( Yarrowia lipolytica ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Escherichia coli ( Escherichia coli ).

9. The recombinant Saccharomyces cerevisiae according to any one of claims 1-8 Saccharomyces cerevisiae Use of the CEN.PK2-1C engineered strain in the preparation of a heme-binding protein or a derivative thereof.

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