Construction and use of pichia pastoris chassis strain for synthesizing highly active heme proteins

Abstract

The present invention relates to the technical field of genetic engineering. Disclosed are construction and use of a pichia pastoris chassis strain for synthesizing highly active heme proteins. In the present invention, a pichia pastoris chassis strain for efficiently synthesizing highly active heme proteins is constructed by enhancing the transcription of globin genes, inhibiting the degradation of globin, and optimizing the supply of heme prosthetic groups. At the shake flask level, the titers of porcine myoglobin, bovine myoglobin, soybean hemoglobin, clover hemoglobin, porcine hemoglobin α subunit, porcine hemoglobin β subunit, Vitreoscilla hemoglobin, and cytochrome P450-BM3 mutant (A82F / A328F) reach 5.02-286.53 mgL-1, and the heme binding rates of these heme proteins reach 13.24%-79.47%. The constructed heme-supplying pichia pastoris chassis strain and strategy in the present invention facilitate synthetization of other highly active heme proteins having a wide application range.

Description

Construction and application of a Pichia pastoris chassis strain for synthesizing highly active heme proteins Technical Field

[0001] The invention relates to the construction and application of a Pichia pastoris chassis strain for synthesizing highly active heme proteins, and belongs to the technical field of genetic engineering. Background Art

[0002] Heme proteins, including hemoglobin, myoglobin, cytochrome P450 enzymes, catalases, and peroxidases, play crucial roles in both prokaryotic and eukaryotic cells, including oxygen transport, electron transfer, gas sensing, signal transduction, circadian clock regulation, and microRNA processing. With the increasing demand for heme proteins, including porcine myoglobin (P-Mb), bovine myoglobin (B-Mb), soy leghemoglobin (S-Hb), clover hemoglobin (C-Hb), porcine hemoglobin (P-Hb), Vitreoscilla hemoglobin (V-Hb), and cytochrome P450-BM3, for the food industry, high-cell-density fermentation, and synthesis of high-value-added compounds, there is a need to develop a microbial platform that can efficiently synthesize these highly bioactive heme proteins.

[0003] Escherichia coli is not suitable for food production due to its high risk of endotoxins. Furthermore, the inefficient secretion expression of Saccharomyces cerevisiae increases purification costs, limiting the industrial-scale production of heme proteins. Pichia pastoris is an FDA-approved and recognized safe host. Its protein expression system, with its significant advantages (high secretion efficiency, low protein glycosylation, and high-density culture), is widely used in the production of recombinant proteins, industrial enzymes, pharmaceuticals, and natural products. Therefore, using the safe host Pichia pastoris expression system to synthesize highly active heme proteins is the best choice.

[0004] Synthesizing heme proteins in Pichia pastoris presents two challenges: first, achieving efficient expression of the globin component of heme proteins; and second, enhancing the intracellular heme supply to meet the heme prosthetic group requirement during heme protein synthesis. The Pichia pastoris system has developed a sophisticated genome-integrated expression system and process control strategies for high-density expression, making this relatively straightforward. While heme supply can be improved through exogenous addition of heme or its key precursor, ALA, the high cost of ALA makes it unsuitable for large-scale industrial production. Furthermore, due to Pichia pastoris' limited capacity for exogenous heme uptake, enhancing intracellular heme biosynthesis is a viable approach for producing highly active heme proteins. However, studies have shown that overexpression of all genes in the heme biosynthesis pathway results in the accumulation of large amounts of heme intermediates rather than heme itself. Therefore, optimizing the expression of heme biosynthetic enzymes rather than simply overexpressing them to improve heme supply is crucial. In Saccharomyces cerevisiae, eight heme biosynthetic enzymes are reportedly distributed in the cytoplasm (Hem2p, Hem3p, Hem4p, Hem12p, and Hem3p) and mitochondria (Hem1p, Hem14p, and Hem15p). During this natural heme biosynthesis process, heme intermediates must cross the inner and outer mitochondrial membranes multiple times, reducing the efficiency of heme synthesis and utilization. Furthermore, Hem2p and Hem3p are rate-limiting steps in yeast heme biosynthesis, and when Hem4p activity is limited, hydroxymethylbilin (HMB) is spontaneously converted to the byproduct uroporphyrinogen I. Therefore, reestablishing the heme biosynthesis pathway in the cytoplasm and eliminating the rate-limiting steps catalyzed by Hem2p, Hem3p, and Hem4p are crucial for the efficient synthesis of heme and heme-like proteins in yeast. Therefore, this patent constructs an effective Pichia pastoris heme supply chassis strain by removing the spatial isolation in the heme biosynthesis process (cytoplasmic localization of Hem14p and Hem15p), optimizing the synthesis of the precursor 5-aminolevulinic acid (screening of C4 and C5 synthesis pathways), eliminating the rate-limiting step (protein scaffold spatial assembly of rate-limiting enzymes Hem2p, Hem3p and Hem4p), alleviating the accumulation of the intermediate coproporphyrinogen III (overexpression of the HEM13 gene) and inhibiting heme degradation (knockout of the heme oxygenase Hmx1p).

[0005] Summary of the Invention

[0006] The present invention provides a genetically engineered bacterium that has been improved upon the original strain by at least one of the following:

[0007] (1) Knockout or silencing of the key gene ku70 (shown in SEQ ID NO. 1) responsible for the non-homologous end joining repair mechanism to improve the efficiency of gene integration or knockout;

[0008] (2) integrating a transcriptional activator or a molecular chaperone into the genome of the starting strain; the transcriptional activator includes Mit1 (shown in SEQ ID NO. 2), Mxr1 (shown in SEQ ID NO. 3), or Prm1 (shown in SEQ ID NO. 4), and the molecular chaperone includes PDI (shown in SEQ ID NO. 5) or KAR2 (shown in SEQ ID NO. 6);

[0009] (3) knocking out one or more of the proteases Pep4 (shown in SEQ ID NO. 7), Prb1 (shown in SEQ ID NO. 8), and Yps1-1 (shown in SEQ ID NO. 9);

[0010] (4) Through bioinformatics analysis and co-expression of mScarlet red fluorescent protein (SEQ ID NO.10), the subcellular localization and possible mitochondrial localization signal peptides of heme biosynthetic enzymes (Hem1p, gene as shown in SEQ ID NO.11; Hem2p, gene as shown in SEQ ID NO.12; Hem3p, gene as shown in SEQ ID NO.13; Hem4p, gene as shown in SEQ ID NO.14; Hem12p, gene as shown in SEQ ID NO.15; Hem13p, gene as shown in SEQ ID NO.16; Hem14p, gene as shown in SEQ ID NO.17; Hem15p, gene as shown in SEQ ID NO.18) were revealed in Pichia pastoris. The mitochondrial localization signal peptides of mitochondrial heme biosynthetic enzymes on the Pichia pastoris genome were specifically knocked out using CRISPR / Cas9 technology to localize them to the cytoplasm for functional expression.

[0011] (5) Introducing the C4 synthesis pathway or the C5 synthesis pathway to improve the 5-aminolevulinic acid synthesis capacity; the gene HEM1 (shown in SEQ ID NO.11) encoding the C4 synthesis pathway is derived from Pichia pastoris, and the gene encoding the C5 synthesis pathway is derived from Escherichia coli (glutamyl tRNA reductase gene GluTR E fbr , shown in SEQ ID NO.19; glutamate-semialdehyde aminotransferase gene GSAM E , shown in SEQ ID NO.20) and Bacillus subtilis (glutamyl tRNA reductase gene GluTR B fbr , shown in SEQ ID NO.21; glutamate-semialdehyde aminotransferase gene GSAM B , shown in SEQ ID NO.22);

[0012] (6) Hem2p, Hem3p, and Hem4p fused with specific ligands (HEM2-linker-GBD ligand, shown in SEQ ID NO. 23; HEM3-linker-SH3 ligand, shown in SEQ ID NO. 24; HEM4-linker-PDZ ligand, shown in SEQ ID NO. 25) were fixed to a protein scaffold containing the metazoan signaling protein interaction domains (GBD, SH3, and PDZ) (GBD domain-linker-SH3 domain-linker-PDZ domain, shown in SEQ ID NO. 26) for spatial assembly to optimize the synthesis of intermediates (porphobilinogen, hydroxymethylbilinogen, and uroporphyrinogen III);

[0013] (7) Use P GAP The promoter (shown in SEQ ID NO. 27) enhances the expression of the HEM13 gene and alleviates the accumulation of the intermediate coproporphyrinogen III;

[0014] (8) Knockout or silence the heme oxygenase HMX1 gene (shown in SEQ ID NO. 28) to inhibit heme degradation.

[0015] In one embodiment, CRISPR / Cas9 technology is used to specifically knock out the key gene ku70 responsible for the non-homologous end joining repair mechanism.

[0016] In one embodiment, the gene encoding the key gene ku70 responsible for the non-homologous end joining repair mechanism is Gene ID: 8199462.

[0017] In one embodiment, the transcriptional activator Mit1, Mxr1 or Prm1, or the molecular chaperone PDI or KAR2 is integrated into the Pichia pastoris genome P AOX1 UP-g2 site.

[0018] In one embodiment, the gene ID encoding the methanol-induced transcription factor 1 gene Mit1 is 8200325; the gene ID encoding the methanol expression regulator 1 gene Mxr1 is 8201433; and the gene ID encoding the methanol utilization pathway gene positive regulator gene Prm1 is 8201109.

[0019] In one embodiment, the protease Pep4, Prb1 or Yps1-1 is specifically knocked out using CRISPR / Cas9 technology.

[0020] In one embodiment, the gene encoding the vacuolar aspartyl protease Pep4 is Gene ID: 8200047; the gene encoding the vacuolar serine protease Prb1 is Gene ID: 8196728; and the gene encoding the GPI-anchored aspartyl protease Yps1-1 is Gene ID: 8196641.

[0021] In one embodiment, the mScarlet red fluorescent protein is fused to a heme biosynthetic enzyme and integrated into the Pichia pastoris genome via an integration expression plasmid. The recombinant strains are then stained with a mitochondrial-specific dye (Mitotracker Green FM) and analyzed by fluorescence microscopy to reveal the subcellular localization of these heme biosynthetic enzymes in Pichia pastoris and possible mitochondrial localization signal peptides. Finally, the CRISPR / Cas9 technology is used to specifically knock out the mitochondrial localization signal peptides of these mitochondrial heme biosynthetic enzymes to localize them to the cytoplasm for functional expression, thereby removing the spatial isolation barrier in the heme synthesis process.

[0022] In one embodiment, the protein ID encoding the red fluorescent protein mScarlet is QBH90981.1; the gene ID encoding the 5-aminolevulinic acid synthase HEM1 is 8198334; the gene ID encoding the porphobilinogen synthase HEM2 is 8200701; the gene ID encoding the porphobilinogen deaminase HEM3 is 8197753; the gene ID encoding the uroporphyrinogen III synthase HEM4 is 8197972; the gene ID encoding the uroporphyrinogen III decarboxylase HEM12 is 8199701; the gene ID encoding the coproporphyrinogen III oxidase HEM13 is 8200294; the gene ID encoding the protoporphyrinogen IX oxidase HEM14 is 8201145; and the gene ID encoding the ferrochelatase HEM15 is 8200092.

[0023] In one embodiment, the red fluorescent protein mScarlet is linked to the heme biosynthesis enzyme via the promoter P GAP Regulate expression.

[0024] In one embodiment, the HEM1 gene is expressed in Pichia pastoris by integrating an expression plasmid, or the gene is integrated into the Pichia pastoris genome by CRISPR / Cas9 technology for overexpression.

[0025] In one embodiment, the genes of the C5 biosynthesis pathway derived from Escherichia coli and Bacillus subtilis are expressed in Pichia pastoris by integrating an expression plasmid.

[0026] In one embodiment, the gene HEM1 and genes from the C5 biosynthesis pathway of Escherichia coli and Bacillus subtilis are expressed via promoter P GAP or P AOX1 (shown in SEQ ID NO.29) to regulate expression.

[0027] In one embodiment, the integration expression vector includes but is not limited to pPICZαA, pPICZαB, pPICZαC or pGAPZαA, pGAPZαB, pGAPZαC.

[0028] In one embodiment, any one of the following genes is expressed in Pichia pastoris by integrating an expression vector: Gene ID: 8198334 encoding Pichia pastoris 5-aminolevulinic acid synthase gene HEM1; Gene ID: 8198334 encoding Escherichia coli glutamyl tRNA reductase gene GluTR E Gene ID: 945777; encoding Escherichia coli glutamate-semialdehyde aminotransferase GSAM E Gene ID: 946892; encoding Bacillus subtilis glutamyl tRNA reductase gene GluTR B Gene ID: 937443; Gene encoding Bacillus subtilis glutamate-semialdehyde aminotransferase GSAM B Gene ID: 937490.

[0029] In one embodiment, CRISPR / Cas9 technology is used to replace the native HEM2, HEM3, and HEM4 genes in the Pichia pastoris genome with the HEM2-linker-GBD ligand, the HEM3-linker-SH3 ligand, and the HEM4-linker-PDZ ligand, respectively.

[0030] In one embodiment, CRISPR / Cas9 technology is also used to replace the constitutive promoter P GAP (medium intensity) or P G7 (Low intensity) driven protein scaffold GBD domain-linker-SH3 domain-linker-PDZ domain integrated into the Pichia pastoris genome P TEF1 UP-g1 site.

[0031] In one embodiment, the gene ID encoding the porphobilinogen synthase gene HEM2 is 8200701; the gene ID encoding the porphobilinogen deaminase gene HEM3 is 8197753; the gene ID encoding the uroporphyrinogen III synthase gene HEM4 is 8197972; the GBD ligand is the amino acid sequence (LVGALMHVMQKRSRAIHSSDEGEDQAGDEDED); the SH3 ligand is the amino acid sequence (PPPALPPKRRR); the PDZ ligand is the amino acid sequence (GVKESLV); the protein ID encoding the GBD domain is BAA21534.1 (residues from 196 to 274); the protein ID encoding the SH3 domain is AAH31149.1 (residues from 133 to 190); the protein ID encoding the PDZ domain is EDL06069.1 (residues from 77 to 171); the protein ID encoding the P G7 The promoter contains the nucleotide sequence shown in SEQ ID NO.30.

[0032] In one embodiment, the natural promoter P of HEM13 on the Pichia pastoris genome is modified using CRISPR / Cas9 technology. HEM13 Replace with P GAP , to enhance the expression of the HEM13 gene and alleviate the accumulation of the intermediate coproporphyrinogen III. In one embodiment, the heme degradation enzyme Hmx1p is specifically knocked out in the Pichia pastoris genome using CRISPR / Cas9 technology.

[0033] In one embodiment, the gene encoding the heme degrading enzyme HMX1 is Gene ID: 8199419.

[0034] In one embodiment, the genetically engineered bacteria further express heme proteins; the heme proteins include porcine myoglobin P-Mb (shown in SEQ ID NO.31), bovine myoglobin B-Mb (shown in SEQ ID NO.32), soy leghemoglobin S-Hb (shown in SEQ ID NO.33), clover hemoglobin C-Hb (shown in SEQ ID NO.34), porcine hemoglobin P-Hb (α subunit shown in SEQ ID NO.35; β subunit shown in SEQ ID NO.36), Vitreoscilla hemoglobin V-Hb (shown in SEQ ID NO.37), wild-type cytochrome P450-BM3 mutant (A82F / A328F, abbreviated as BM3 mut , shown in SEQ ID NO.38).

[0035] In one embodiment, the Gene ID encoding porcine myoglobin gene P-Mb is 397467; the Gene ID encoding bovine myoglobin gene B-Mb is 280695; the Gene ID encoding soybean hemoglobin gene S-Hb is 100527379; the Protein ID encoding clover hemoglobin C-Hb is GAU42437.1; the Gene ID encoding porcine hemoglobin α subunit P-Hbα is 110259958; the Gene ID encoding porcine hemoglobin β subunit P-Hbβ is 407066; the Protein ID encoding Vitreoscilla hemoglobin V-Hb is WP_019959060.1; and the Protein ID encoding wild-type cytochrome P450-BM3 is WP_034650526.1.

[0036] In one embodiment, the genetically engineered bacteria uses Pichia pastoris or Saccharomyces uvarum as a host.

[0037] The present invention also provides a method for fermenting and producing heme and / or heme-like proteins, wherein the method uses the genetically engineered bacteria as a fermentation microorganism, inoculates the fermentation medium with a bacterial concentration of 1×10 5 ~1×10 9 CFU / mL of bacterial cells, ferment at 25-35°C, 100-600 rpm for at least 24 h.

[0038] In one embodiment, the fermentation medium includes but is not limited to YNB, YPD, BMGY or BMMY medium.

[0039] In one embodiment, the fermentation medium further contains essential amino acids required by the bacteria.

[0040] In one embodiment, feeding is also performed during the fermentation process.

[0041] The present invention also provides a method for producing ALA by fermentation, wherein the method uses the genetically engineered bacteria as a fermentation microorganism, inoculates the fermentation medium with a bacterial concentration of 1×10 5 ~1×10 9 CFU / mL of bacterial cells, ferment at 25-35°C, 100-600 rpm for at least 24 h.

[0042] In one embodiment, the fermentation medium includes but is not limited to YNB, YPD, BMGY or BMMY medium.

[0043] In one embodiment, the fermentation medium further contains essential amino acids required by the bacteria.

[0044] The present invention also provides the use of the genetically engineered bacteria as chassis cells in the development of artificial meat, high cell density fermentation, or whole-cell catalytic synthesis of high-value-added compounds.

[0045] Beneficial effects: The present invention constructs a Pichia pastoris chassis strain for efficiently synthesizing highly active heme proteins, which is based on enhanced globin gene transcription, inhibited globin degradation and optimized heme prosthetic group supply (cytoplasmic localization of Hem14p and Hem15p, enhanced precursor ALA synthesis, protein scaffold assembly of rate-limiting enzymes Hem2p, Hem3p and Hem4p, overexpression of HEM13 gene to alleviate the accumulation of intermediate CPG III and inhibit heme degradation). At the shake flask level, porcine myoglobin (P-Mb), bovine myoglobin (B-Mb), soy leghemoglobin (S-Hb), clover hemoglobin (C-Hb), porcine hemoglobin α subunit (P-Hbα), porcine hemoglobin β subunit (P-Hbβ), Vitreoscilla hemoglobin (V-Hb) and cytochrome P450-BM3 mutants (A82F / A328F, BM3 mut ) reached 246.95, 20.04, 286.53, 5.02, 10.16, 5.24, 66.30, and 57.27 mg L -1 Furthermore, the heme-binding rates of these heme proteins reached 60.02%, 40.15%, 79.47%, 15.46%, 18.27%, 13.24%, 25.94%, and 61.27%, respectively. Therefore, the efficient heme-supplying Pichia pastoris chassis strain and strategy developed here will facilitate the synthesis of other highly active heme proteins with broad applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 shows the selection of a suitable expression system for the globin component in P-Mb; lanes 1 to 4 are X33-P AOX1 -α-(P-Mb) cn＝1 、KM71-P AOX1 -α-(P-Mb) cn＝1 、SMD1168-P AOX1 -α-(P-Mb) cn＝1 With GS115-P AOX1 -α-(P-Mb) cn＝1 The supernatant of the strain after 48 hours of fermentation; lanes 5 to 7 are X33-P AOX1 -α-(P-Mb) cn＝1 (single copy strain), X33-P AOX1 -α-(P-Mb) cn＝2 (Dual copy strain) and X33-P AOX1 -α-(P-Mb) cn＝3(Three-copy strain) fermentation supernatant at 48h; lanes 8 to 11 are X33-P AOX1 -α-(P-Mb) cn＝1 The supernatants of the strains at 24, 48, 72 and 96 h of fermentation; lanes 12 to 15 are X33-P AOX1 -α-(P-Mb) cn＝1 Cell lysates of the strain at 24, 48, 72, and 96 hours of incubation; Lane 16 shows cell lysates of the X33 strain lacking the P-Mb gene at 48 hours of incubation. ExPASy predicted the molecular weight of the 6×His-tagged P-Mb to be 17.91 kDa. M represents protein standard; cn represents copy number. Data are presented as mean ± SD of three independent biological replicates (n = 3).

[0047] Figure 2 shows CRISPR / Cas9-mediated ku70 knockout; a is the double-strand break introduced by Cas9 cleavage, which is mainly repaired by homology-directed repair in Δku70 but mainly by non-homologous end joining repair in the wild type; b is a schematic diagram of the CRISPR / Cas9 plasmid used to knock out the ku70 gene. The RGR operon consists of the gRNA and its two flanking self-cleaving ribonucleases (HH and HDV), which are regulated by the bidirectional promoter P HTX1 control. PARS, autonomously replicating sequence from P. pastoris X33. Lanes 1 to 3 represent X33-Δku70, X33-ku70, and DNA marker, respectively.

[0048] Figure 3 shows the changes in P-Mb titer after overexpression of molecular chaperones and transcription activators; lanes 1 to 7 are X33-Δku70-P GAP -PDI-(P-Mb), X33-Δku70-P GAP -KAR2-(P-Mb), X33-Δku70-P GAP -Mxr1-(P-Mb), X33-Δku70-P GAP -Mit1-(P-Mb), X33-Δku70-P GAP 48-hour fermentation supernatants of Prm1-(P-Mb), X33-Δku70-(P-Mb), and X33-Δku70 strains. M represents protein standard, and CK represents the control strain X33-Δku70-(P-Mb). Data are presented as mean ± SD of three independent biological replicates (n = 3). Statistical comparison (p-value) was performed using a two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001, NS indicates not significant (p ≥ 0.05).

[0049] Figure 4 shows the changes in P-Mb titer after protease knockout; lanes 1 to 6 represent the 48-h fermentation supernatants of strains X33-Δku70-Δpep4-(P-Mb), X33-Δku70-Δyps1-(P-Mb), X33-Δku70-Δprb1-(P-Mb), X33-Δku70-Δpep4-Δyps1-(P-Mb), X33-Δku70-Δpep4-Δyps1-Δprb1-(P-Mb), and X33-Δku70-(P-Mb), respectively. M represents protein standard, and CK represents the control strain X33-Δku70-(P-Mb). Data are presented as mean ± SD of three independent biological replicates (n = 3). Statistical evaluation (p-value) compared to the control was performed using a two-tailed t-test. *p<0.05, **p<0.01, ***p<0.001, NS means not significant (p≥0.05).

[0050] Figure 5 shows the secretory expression of P-Mb and S-Hb in the P1 strain, as well as the secretory expression of V-Hb and BM3 mut Lanes 1 to 2 are the fermentation supernatant and purified samples of the P1-(P-Mb) strain at 48 hours; lanes 3 to 4 are the fermentation supernatant and purified samples of the P1-(S-Hb) strain at 48 hours; lanes 5 to 6 are the cell lysate and purified samples of the P1-(V-Hb) strain at 48 hours; lanes 7 to 8 are the cell lysate and purified samples of the P1-BM3 mut Cell lysate and purified samples of the strain at 48 hours; Lane 9 is the cell lysate of the P1 strain at 48 hours. ExPASy predicted P-Mb, S-Hb, V-Hb and BM3 containing 6×His tags mut The molecular weights of V-Hb are 17.91, 16.35, 16.60, and 118.76 kDa, respectively. V-Hb is a homodimer composed of two identical subunits and two heme molecules, resulting in a molecular weight of 33.20 kDa. Data are presented as mean ± standard deviation of three independent biological replicates (n = 3).

[0051] Figure 6 shows the reconstruction of the heme biosynthesis pathway in the cytoplasm. a is a schematic diagram of the subcellular localization of the heme biosynthetic enzyme (HBS) and its potential mitochondrial localization signal peptide (MLS); b is the analysis of the intracellular localization of the native HBS by fluorescence microscopy, CK represents the recombinant strain expressing HBS fused with m-Scarlet observed in the same field of view using white light; c is the subcellular localization of the N-terminal truncated versions of Hem1p, Hem14p, and Hem15p; d is the subcellular localization of HEM14 71-561 and HEM15 71-375Effects of cytoplasmic coexpression on cell growth and heme synthesis. CK represents the control strain X33-Δku70. Data are presented as the mean ± standard deviation of three independent biological replicates (n = 3). Statistical evaluation (p-value) was performed using a two-tailed t-test compared with the control strain. *p < 0.05, **p < 0.01, ***p < 0.001, NS represents nonsignificant (p ≥ 0.05). Abbreviations: mSca, m-Scarlet; MTG, Mitotracker Green FM; aa, amino acid.

[0052] Figure 7 shows the moderate enhancement effect of the reconstructed heme biosynthesis pathway; wherein, a is the key step of multi-enzyme assembly to enhance heme biosynthesis (Hem2p, Hem3p and Hem4p). Hem2p, Hem3p and Hem4p fused with specific ligands are fixed to the constitutive promoter P GAP (medium strength, HEME-4 strain) and P G7 (Weak, HEME-5 strain) driven by a protein scaffold. The HEME-3 strain, which lacks a synthetic protein scaffold, served as a control. Scaf, scaffold; Lig, ligand; Dom, domain. b. Effects of overexpressing HEM13 (HEME-7 strain) and deleting HMX1 (HEME-8 strain) on intracellular heme accumulation, with HEME-6 serving as a control strain. c. Comparison of heme biosynthesis between the final heme-supply strain HEME-9 and the original strain X33-Δku70.

[0053] Figure 8 shows the biochemical characteristics of biosynthetic heme proteins; wherein, a represents the expression of several heme proteins in the P1H9 strain, lanes 1 to 2 represent the fermentation supernatant and purified samples of the P1H9-(P-Mb) strain at 48 h; lanes 3 to 4 represent the fermentation supernatant and purified samples of the P1H9-(S-Hb) strain at 48 h; lanes 5 to 6 represent the cell lysate and purified samples of the P1H9-(V-Hb) strain at 48 h; lanes 7 to 8 represent the cell lysate and purified samples of the P1H9-BM3 mut Cell lysate and purified samples of the strain at 48 hours; Lane 9 is the cell lysate of the P1H9 strain at 48 hours; M represents the protein standard; b is the spectral characteristics of the purified heme protein; c is the heme binding rate of the purified heme protein detected by the difference spectrum between the reduced and oxidized samples; d is the specific peroxidase activity of purified P-Mb and S-Hb; e is the time course of the oxygen binding behavior of V-Hb in the presence of sodium dithionite (reducing agent and oxygen scavenger); S at.O2 represents oxygen saturation; f represents the expression of P450-BM3 mut The catalytic efficiency of the cells in converting phenol to hydroquinone was analyzed by HPLC.

[0054] Figure 9 shows the secretory expression of B-Mb, C-Hb, P-Hbα, and P-Hbβ in the P1H9 strain. The molecular weights of B-Mb, C-Hb, P-Hbα, and P-Hbβ containing a 6×His tag predicted by ExPASy are 17.90, 16.51, 15.99, and 16.99 kDa, respectively. Data are presented as the mean ± standard deviation of three independent biological replicates (n=3). Lanes 1 to 4 are P1H9-P AOX1 -α-(B-Mb) strain fermentation supernatants at 12, 24, 36, and 48 h; lanes 5 to 6 are P1H9-P AOX1 -α-(C-Hb) strain fermentation supernatant at 24 and 48 h; lanes 7 to 8 are P1H9-P AOX1 -α-(P-Hbα) and P1H9-P AOX1 -α-(P-Hbβ) strain fermentation supernatant at 48 h; M represents protein standard. DETAILED DESCRIPTION

[0055] Determination of 5-aminolevulinic acid: HPLC analysis was performed using OPA (o-phthalaldehyde) boric acid pre-column derivatization. (1) Mobile phase: Phase A, 3.01 g of anhydrous sodium acetate, 200 μL of triethylamine, dilute to 1 L, adjust the pH to 7.20 ± 0.05 with 5% acetic acid, filter (water system) and add 5 mL of tetrahydrofuran; Phase B, 3.01 g of anhydrous sodium acetate, dilute to 200 mL, adjust the pH to 7.20 ± 0.05 with 5% acetic acid, filter (water system) and add 400 mL of methanol and 400 mL of acetonitrile; (2) Chromatographic column: C18 column (5 μm, 4.6 mm × 250 mm, Agilent, USA), column temperature 40 ° C, detection wavelength: 338 nm; flow rate: 1.0 mL / min; injection volume: 20 μL; (3) injection procedure: ① extraction, using the default compensation value to extract 7 μL from the position "P1-A-4" containing 200 μL / min; ② cleaning, cleaning the needle once at the position "P1-A-3"; ③ extraction, using the default compensation value to extract 1 μL from the sample containing 200 μL / min; ④ cleaning, cleaning the needle once at the position "P1-A-3"; ⑤ mixing, mixing 8 μL from the air with the maximum speed 3 times; ⑥ extraction, using the default compensation value to extract 2 μL from the position "P1-A-1" containing 200 μL / min; ⑦ Wash, wash the needle once at position "P1-A-3"; ⑧ Mix, mix 10 μL from the air at maximum speed 15 times; ⑨ Draw, draw 30 μL from position "P1-A-5" at 200 μL / min using the default compensation value; ⑩ Mix, mix 20 μL from the air at 300 μL / min 3 times; Injection. Position: "P1-A-1", OPA derivatization reagent; "P1-A-3 and P1-A-5", ultrapure water; "P1-A-4", boric acid.

[0056] (5) Gradient elution:

[0057] Table 1 Elution program settings

[0058] Hemoglobin detection method: The concentration of intracellular hemoglobin was determined by oxalic acid extraction method. 600 ×mL=8 cells were placed in a brown EP tube, centrifuged at 12000rpm for 5min to collect the bacteria, and then washed twice with PBS buffer (pH 7.4). After centrifugation, the supernatant was discarded, 500μL of 20mM oxalic acid was added to the bacteria and mixed, and placed at 4°C for 16h. Next, the mixture was mixed with 500μL of 2M oxalic acid. After mixing, 500μL of the system was taken out as a control, and the other half was incubated at 95°C for 30min. After incubation, the extract was centrifuged at 12000rpm for 5min, and the supernatant was used for fluorescence measurement. Fluorescence was detected using a microplate reader (BioTek Synergy H1) at excitation λ=400nm and emission λ=600nm. Hemin (Aladdin reagent) was used as a standard for estimating hemoglobin concentration.

[0059] Quantification of heme proteins: Cell growth was monitored by measuring the optical density at 600 nm using a UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan). The secreted P-Mb, S-Hb, B-Mb, C-Hb, P-Hbα, and P-Hbβ in the fermentation supernatant were identified by 10% (W / V) SDS-PAGE. The titers of P-Mb, S-Hb, B-Mb, C-Hb, P-Hbα, and P-Hbβ were quantitatively analyzed by combining the Bradford method (P0006, Beyotime Biotech, Shanghai, China) and Quantity-One (Bio-Rad, California, USA). In order to detect the intracellular synthesis of V-Hb and BM3 mut The cell pellets of 50 mL of fermentation sample were broken up using a high-pressure homogenizer (UH-06, Union-Biotech, Shanghai, China), and the corresponding supernatant was purified and analyzed by SDS-PAGE, Braford, and Quantity-One. The purification of secreted or intracellularly synthesized heme proteins was performed using BeaverBeads. TMHis-tag Protein Purification (70501, Beaver Biomedical Engineering, Suzhou, China) was used. The purified blood protein was desalted using Amicon Ultra 3K (Millipore, Darmstadt, Germany).

[0060] Table 2 sgRNA sequences involved in the examples

[0061] Note: The underlined sequence is the PAM sequence.

[0062] The integration site P involved in the following examples AOX1 UP-g2, P TEF1 UP-g1 and AOXTTDOWN are both well-known sites in the art and disclosed in the paper "CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris".

[0063] Example 1 Construction of a suitable expression system for the globin component of heme proteins

[0064] 1) The P-Mb gene fragment (shown in SEQ ID NO. 31) was obtained by PCR amplification and then inserted into the pPICZαA plasmid backbone using Gibson assembly to generate the recombinant plasmid pPICZαA-(P-Mb) cn＝1 Then pPICZαA-(P-Mb) cn＝1 The effect of host type on P-Mb expression was investigated by electroporation and integration into the 5′AOX1 site of the genome of different Pichia pastoris (X33, KM71, SMD1168 and GS115).

[0065] 2) Digestion and assembly of pPICZαA-(P-Mb) with the tailing enzymes Bgl II and BamH I (Invitrogen manual) cn＝1 , producing multimeric pPICZαA-(P-Mb) cn＝2 with pPICZαA-(P-Mb) cn＝3 Then, the recombinant plasmid pPICZαA-(P-Mb) cn＝2 with pPICZαA-(P-Mb) cn＝3 The P-Mb gene was integrated into the 5′AOX1 site of the Pichia pastoris X33 genome to generate Pichia pastoris strains with two and three copies of the P-Mb gene, and the effect of gene copy number on P-Mb expression was investigated.

[0066] 3) The recombinant plasmid pPICZαA-(P-Mb) cn＝1 The protein was integrated into the 5′AOX1 site of the Pichia pastoris X33 genome, and the effect of fermentation time (24 h, 48 h, 72 h and 96 h) on P-Mb expression was investigated.

[0067] Fermentation conditions: Single colonies of these recombinant Pichia pastoris strains were inoculated into 250 mL shake flasks containing 50 mL YPD medium (10 g / L yeast powder, 20 g / L peptone, 2% (w / v) glucose) and shaken at 250 rpm at 30°C until the logarithmic phase (OD 600 =2-6). Next, the seed culture medium was centrifuged at 3000×g for 5 minutes, and the supernatant was discarded. Subsequently, the cell pellet was resuspended twice with physiological saline to remove residual sugars. Finally, the treated cells were transferred to a 250 mL shake flask and 50 mL of BMMY medium (1% (v / v) methanol, 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L YNB, 4 mg / L biotin, 100 mM potassium phosphate buffer, pH 6.0) was added and shaken at 250 rpm at 30°C; methanol was supplemented every 24 hours to a final concentration of 1%.

[0068] Fermentation results: As shown in Figure 1, P-Mb was based on the P. pastoris X33 host. AOX1 The regulated expression of the promoter could achieve a high P-Mb yield (70.76±8.15mg L -1 ) ( FIG1 ). Furthermore, analysis of cell lysates at 24, 48, 72, and 96 hours showed that P-Mb did not accumulate within the cells, indicating that the secretion of P-Mb mediated by the α-factor signal peptide was efficient ( FIG1 ).

[0069] Example 2 Construction of Pichia pastoris X33-Δku70 strain

[0070] In contrast to Saccharomyces cerevisiae, nonhomologous end joining (NHEJ) predominates over homologous recombination (HR) in Pichia pastoris. Knockout of ku70 (SEQ ID NO. 1), a key gene responsible for NHEJ, can improve HR efficiency. Using the genome of Pichia pastoris strain X33 as a template, 1000-bp upstream and downstream fragments of the ku70 gene (Gene ID: 8199462) were amplified. The two fragments were ligated using fusion PCR to construct a knockout cassette. The ku70 gene was then knocked out in Pichia pastoris strain X33 using CRISPR / Cas9 technology. After colony PCR verification and sequencing, the correct knockout strain, X33-Δku70, was obtained (Figure 2).

[0071] Example 3 Overexpression of P AOX1 Transcription activators and molecular chaperones

[0072] Pichia pastoris genomic DNA and pGAPZαA plasmid were used as templates to amplify P AOX1 Upstream and downstream homology arms of UP-g2 (Ag2) site (1kb), P GAP The promoter (shown in SEQ ID NO.27), the transcription activator gene Mit1 (shown in SEQ ID NO.2) and the AOX1 terminator (AOX1TT) were connected by overlapping extension PCR to construct the knock-in cassette UpAg2-P GAP -Mit1-AOX1TT-DoAg2. Then, the knock-in frame was integrated into the Ag2 site of the Pichia pastoris X33-Δku70 genome using CRISPR / Cas9 technology to obtain the recombinant strain X33-Δku70-P GAP In the same manner, the transcriptional activator and the molecular chaperone genes Mxr1 (shown in SEQ ID NO.3), Prm1 (shown in SEQ ID NO.4), PDI (shown in SEQ ID NO.5) and KAR2 (shown in SEQ ID NO.6) were integrated into the Ag2 site of the Pichia pastoris X33-Δku70 genome to obtain the recombinant strains X33-Δku70-P GAP -Mxr1、X33-Δku70-P GAP -Prm1、X33-Δku70-P GAP -PDI and X33-Δku70-P GAP Then, the porcine myoglobin (P-Mb) gene shown in SEQ ID NO.31 was integrated into the 5′AOX1 site (upstream 5′ end of the AOX1 gene) of the transcription activator and molecular chaperone overexpression strain using the integration expression plasmid pPICZαA to obtain the recombinant strains X33-Δku70-P GAP -Mit1-(P-Mb), X33-Δku70-P GAP -Mxr1-(P-Mb), X33-Δku70-P GAP -Prm1-(P-Mb), X33-Δku70-P GAP -PDI-(P-Mb) and X33-Δku70-P GAP -KAR2-(P-Mb).

[0073] Fermentation conditions: Single colonies of these recombinant Pichia pastoris strains were inoculated into 250 mL shake flasks containing 50 mL YPD medium (10 g / L yeast powder, 20 g / L peptone, 2% (w / v) glucose) and shaken at 250 rpm at 30°C until the logarithmic phase (OD 600 =2-6). Next, the seed culture medium was centrifuged at 3000×g for 5 minutes, and the supernatant was discarded. Subsequently, the cell pellet was resuspended twice with physiological saline to remove residual sugars. Finally, the treated cells were transferred to a 250 mL shake flask and 50 mL of BMMY medium (1% (v / v) methanol, 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L YNB, 4 mg / L biotin, 100 mM potassium phosphate buffer, pH 6.0) was added. The flask was shaken at 250 rpm at 30°C for 48 hours; methanol was supplemented every 24 hours to a final concentration of 1%.

[0074] Fermentation results: X33-Δku70-P GAP The -Mit1-(P-Mb) strain had the highest P-Mb production, reaching 128.65±8.27 mg / L, which was 74.37% higher than that of the control strain X33-Δku70 ( FIG3 ).

[0075] Example 4 Knockout of proteases Pep4, Prb1 and Yps1-1

[0076] 1) Using the genome of the Pichia pastoris strain X33-Δku70 constructed in Example 2 as a template, the upstream and downstream homology arms (1 kb) of the protease pep4 (shown in SEQ ID NO. 7), prb1 (shown in SEQ ID NO. 8), and yps1-1 (shown in SEQ ID NO. 9) genes were amplified respectively. The two fragments were connected by overlap extension PCR to construct knockout cassettes of the corresponding three protease genes. Then, the pep4, prb1, and yps1-1 genes (pep4, Gene ID: 8200047; prb1, Gene ID: 8196728; yps1-1, Gene ID: 8196641) were knocked out in the Pichia pastoris X33-Δku70 strain respectively by CRISPR / Cas9 technology to obtain protease single knockout strains X33-Δku70-Δpep4, X33-Δku70-Δyps1, and X33-Δku70-Δprb1.

[0077] 2) Construction of a Pichia pastoris protease double knockout strain: According to the above method, the yps1-1 gene in the Pichia pastoris strain X33-Δku70-Δpep4 was knocked out using CRISPR / Cas9 technology to obtain the protease double knockout strain X33-Δku70-Δpep4-Δyps1.

[0078] 3) Construction of a Pichia pastoris protease triple knockout strain: According to the above method, the prb1 gene in the Pichia pastoris strain X33-Δku70-Δpep4-Δyps1 was knocked out using CRISPR / Cas9 technology to obtain the protease triple knockout strain X33-Δku70-Δpep4-Δyps1-Δprb1.

[0079] 4) The porcine myoglobin (P-Mb) gene was integrated into the 5′AOX1 site (5′ upstream of the AOX1 gene) on the genome of the protease single-knockout, double-knockout and triple-knockout strains using the integration expression plasmid pPICZαA to obtain the recombinant strains X33-Δku70-Δpep4-(P-Mb), X33-Δku70-Δyps1-(P-Mb), X33-Δku70-Δprb1-(P-Mb), X33-Δku70-Δpep4-Δyps1-(P-Mb) and X33-Δku70-Δpep4-Δyps1-Δprb1-(P-Mb).

[0080] Fermentation conditions: Single colonies of these recombinant Pichia pastoris strains were inoculated into 250 mL shake flasks containing 50 mL YPD medium (10 g / L yeast powder, 20 g / L peptone, 2% (w / v) glucose) and shaken at 250 rpm at 30°C until the logarithmic phase (OD 600 =2-6). Next, the seed culture medium was centrifuged at 3000×g for 5 minutes, and the supernatant was discarded. Subsequently, the cell pellet was resuspended twice with physiological saline to remove residual sugars. Finally, the treated cells were transferred to a 250 mL shake flask and 50 mL of BMMY medium (1% (v / v) methanol, 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L YNB, 4 mg / L biotin, 100 mM potassium phosphate buffer, pH 6.0) was added. The flask was shaken at 250 rpm at 30°C for 48 hours; methanol was supplemented every 24 hours to a final concentration of 1%.

[0081] Fermentation results: Among the five protease-deficient strains (Δpep4, Δyps1, Δprb1, Δpep4-Δyps1, and Δpep4-Δyps1-Δprb1), the X33-Δku70-Δyps1-(P-Mb) strain had the highest P-Mb yield of 117.67 ± 15.24 mg L -1 , which was 56.79% higher than that of the control strain X33-Δku70 ( FIG4 ).

[0082] Example 5 Construction of an Optimal Expression Platform for Globin Components in Heme Proteins

[0083] The Mit1 gene was integrated into the X33-Δku70-Δyps1 strain to construct the optimal platform for globin expression (P1 strain). In the P1 strain, P-Mb achieved the highest yield of 162.46 ± 11.12 mg L -1 (Figure 5), compared with X33-Δku70-P GAP -Mit1-(P-Mb) and X33-Δku70-Δyps1-(P-Mb) strains were 26.28% and 38.06% higher, respectively. Considering the excellent ability of P1 strain to express globin efficiently, we also tried the secretory expression of S-Hb and the secretory expression of V-Hb and BM3 mut The results showed that the P1 strain is also suitable for expressing S-Hb, V-Hb and BM3. mut , and their yields were 184.35±17.14 mg L -1 , 32.32±5.94mg L -1 and 28.31 ± 5.44 mg L -1 (Figure 5).

[0084] Example 6 Bioinformatics Analysis and Fluorescent Protein Co-expression Reveal the Subcellular Localization of Heme Synthase in Pichia pastoris

[0085] 1) The mScarlet gene (shown in SEQ ID NO.10) was sent to Nanjing GenScript for yeast codon optimization and subcloned into the pUC57 vector to generate the pUC57-mScarlet plasmid. Using the Pichia pastoris genome and the synthesized pUC57-mScarlet plasmid as templates, PCR amplification was performed to obtain the following gene fragments: HEM1 (shown in SEQ ID NO.11), HEM2 (shown in SEQ ID NO.12), HEM3 (shown in SEQ ID NO.13), HEM4 (shown in SEQ ID NO.14), HEM12 (shown in SEQ ID NO.15), HEM13 (shown in SEQ ID NO.16), HEM14 (shown in SEQ ID NO.17), HEM15 (shown in SEQ ID NO.18), and mScarlet (shown in SEQ ID NO.10).

[0086] 2) Insert the fragment from step 1) into the multiple cloning site of the pGAPZA plasmid using Gibson assembly to obtain recombinant plasmids pGAPZA-HEM1-linker-mScarlet, pGAPZA-HEM2-linker-mScarlet, pGAPZA-HEM3-linker-mScarlet, pGAPZA-HEM4-linker-mScarlet, pGAPZA-HEM12-linker-mScarlet, pGAPZA-HEM13-linker-mScarlet, pGAPZA-HEM14-linker-mScarlet, and pGAPZA-HEM15-linker-mScarlet; the linker is composed of the flexible amino acids Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala.

[0087] 3) The recombinant plasmid obtained in step 2) was linearized and integrated into the 5′ GAP site (5′ end upstream of the GAP gene) on the genome of Pichia pastoris X33-Δku70 to construct the recombinant Pichia pastoris strain X33-Δku70-P GAP -HEM1-linker-mScarlet、X33-Δku70-P GAP -HEM2-linker-mScarlet、X33-Δku70-P GAP -HEM3-linker-mScarlet、X33-Δku70-P GAP -HEM4-linker-mScarlet、X33-Δku70-P GAP -HEM12-linker-mScarlet、X33-Δku70-P GAP -HEM13-linker-mScarlet、X33-Δku70-P GAP -HEM14-linker-mScarlet and X33-Δku70-P GAP -HEM15-linker-mScarlet.

[0088] 4) The recombinant Pichia pastoris strains were stained with the mitochondrial-specific fluorescent dye Mitotracker Green FM and observed under a fluorescence microscope to obtain the subcellular localization of all heme synthases (HBS): Hem1p, Hem14p, and Hem15p were located in the mitochondria, while Hem2p, Hem3p, Hem4p, Hem12p, and Hem13p were located in the cytoplasm ( Figure 6b ).

[0089] 5) Next, the MLS of Hem1p, Hem14p, and Hem15p were predicted using a bioinformatics tool (TPpred 3.0, https: / / tppred3.biocomp.unibo.it) to predict the migration of these three mitochondrial enzymes into the cytoplasm. However, only the MLS Hem1p It can be predicted that (52 residues located at the N-terminus). As shown in steps 1), 2) and 3), a series of N-terminus truncated mutants of Hem1p, Hem14p and Hem15p (Hem1p 53-561aa ;Hem14p 21-561aa 、Hem14p 41-561aa 、Hem14p 61-561aa 、Hem14p 71-561aa with Hem14p 81-561aa ;Hem15p 21-375aa 、Hem15p 41-375aa 、Hem15p 61-375aa 、Hem15p 71-375aa and Hem15p 81-375aa ) were fused with m-Scarlet (via Linker Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala). 53-561aa It indicates that the first 52 amino acids at the N-terminus are truncated and the amino acids at positions 53-561 at the N-terminus are retained. The same applies to the other truncation mutants.

[0090] With Hem1p, Hem14p 61-561aa and Hem15p 61-375aa Compared with the localization of Hem1p 53-561aa 、Hem14p 71-561aa and Hem15p 71-375aa The subcellular localization of MLS was significantly changed from mitochondria to cytoplasm (Figure 6c). Hem1p The MLS between the N-terminal residues 61 and 70 is consistent with the prediction. Hem14p and MLS Hem15p It was also confirmed for the first time in P. pastoris.

[0091] Example 7 Reconstruction of the heme biosynthesis pathway in the cytoplasm

[0092] Based on the detailed information of the subcellular localization and MLS of the heme biosynthetic enzymes, we attempted to reconstruct the heme biosynthetic pathway in the cytoplasm. First, we removed the MLS of the C4 pathway Hem1p or introduced alternative ALA biosynthetic enzymes from the bacterial C5 pathway, including the glutamyl tRNA reductase gene GluTR from Escherichia coli. E fbr , shown in SEQ ID NO.19; glutamate-semialdehyde aminotransferase gene GSAM E , shown in SEQ ID NO.20) and Bacillus subtilis (glutamyl tRNA reductase gene GluTR B fbr , shown in SEQ ID NO.21; glutamate-semialdehyde aminotransferase gene GSAM B , shown in SEQ ID NO.22), attempting to transfer ALA synthesis from mitochondria to the cytoplasm. Construction of the C4 synthesis pathway of 5-aminolevulinic acid: Using the genome of Pichia pastoris as a template, the HEM1 gene (Gene ID: 8198334) and the MLS truncated version of the HEM1 gene were amplified. 53-561 , Gibson Assembly was used to construct integrated expression P GAP -HEM1, P AOX1 -HEM1, P GAP -HEM1 53-561 With P AOX1 -HEM1 53-561 ; Construction of the C5 biosynthesis pathway of 5-aminolevulinic acid: Using the genome of Escherichia coli or Bacillus subtilis as a template, amplify the GluTR E fbr GSAM E GluTR B fbr With GSAM B Gene, Gibson Assembly was used to construct the integration expression vector P GAP -GluTR E fbr -P GAP -GSAM E 、P GAP -GluTR B fbr -P GAP -GSAM B 、P AOX1 -GluTR E fbr -P AOX1 -GSAM E 、P AOX1 -GluTR Bfbr -P AOX1 -GSAM B . The above plasmids were transformed into the 5'GAP (5' end of the GAP gene) or 5'AOX1 site (5' end of the AOX1 gene) on the Pichia pastoris X33-Δku70 genome. Single colonies of these Pichia pastoris recombinant strains were inoculated into 50 mL culture tubes containing 5 mL of YPD liquid medium and cultured overnight at 30°C and 250 rpm. Subsequently, 0.1 mL of the overnight culture was inoculated into a 250 mL shake flask containing 50 mL of YPD medium and fermented at 30°C and 250 rpm for 48 hours. The fermentation results showed that the titers of ALA in these engineered strains were extremely low (Table 3). In contrast, the control strain P GAP -HEM1 and P AOX1 -HEM1 exhibited efficient ALA synthesis, with yields of 100.09 ± 1.29 mg L -1 and 57.70 ± 8.80 mg L -1 . This phenomenon may be due to the fact that the substrate of Hem1p (succinyl-CoA) is mainly present in the mitochondria, making it difficult to be utilized by the MLS-truncated Hem1p in the cytoplasm. In addition, the low expression level limits the application of ALA biosynthetic enzymes derived from these bacteria to synthesize heme in P. pastoris. Therefore, the synthesis of ALA in mitochondria should be maintained during the reconstruction of the heme biosynthetic pathway. In order to promote the accumulation of ALA, our previous study investigated the effect of multi-copy integration expression of the HEM1 gene on ALA synthesis in S. cerevisiae. However, the results showed that there was no significant difference in intracellular heme levels between low-copy and high-copy expression of HEM1. Therefore, a native HEM1 (P GAP -HEM1) expression cassette, resulting in the HEME-1 strain.

[0093] Next, the MLS was amplified using the genome of Pichia pastoris strain X33-Δku70 as a template. Hem14p With MLS Hem15p The 1000bp fragments upstream and downstream of the gene sequence were connected using fusion PCR technology to construct the knockout cassette, UpMLS Hem14p -DoMLS Hem14p With UpMLS Hem15p -DoMLS Hem15p Then, CRISPR / Cas9 technology was used to knock out the MLS on the X33-Δku70 genome. Hem14p With MLS Hem15pAfter colony PCR verification and sequencing, the correct knockout strain HEME-2 was obtained. Single colonies of HEME-2 and X33-Δku70 strains were inoculated into 50 mL culture tubes containing 5 mL YPD liquid medium and cultured overnight at 30°C and 250 rpm. Subsequently, 0.1 mL of the overnight culture was inoculated into 250 mL shake flasks containing 50 mL YPD medium (with or without 150 mg L -1 ALA), fermented at 30℃ and 250rpm for 48h. 71-561 and HEM15 71-375 Co-expression of the genes could synthesize sufficient heme in the cytoplasm to maintain cell growth without the need for ALA supplementation (Figure 6d). -1 In the case of ALA, the intracellular hemoglobin titer of HEME-2 strain was significantly increased (41.94%) compared with the control strain X33-Δku70. This result shows that Hem14p 71-561aa -Hem15p 71-375aa Its substrates (protoporphyrinogen IX and protoporphyrin IX) can be more efficiently utilized to synthesize heme ( Figure 6 d).

[0094] Table 3 Reconstruction of the ALA synthesis pathway in the cytoplasm

[0095] Example 8: Protein Scaffold Spatial Assembly of Rate-Limiting Enzymes Hem2p, Hem3p, and Hem4p

[0096] The native HEM2 (Gene ID: 8200701), HEM3 (Gene ID: 8197753), and HEM4 (Gene ID: 8197972) genes on the Pichia pastoris X33-Δku70 genome were replaced with HEM2-linker-GBD ligand (shown in SEQ ID NO. 23), HEM3-linker-SH3 ligand (shown in SEQ ID NO. 24), and HEM4-linker-PDZ ligand (shown in SEQ ID NO. 25), respectively, to generate the control strain HEME-3. GAP Promoter (medium strength) or P G7 The protein scaffold GBD domain-linker-SH3 domain-linker-PDZ domain (shown in SEQ ID NO.26) driven by the promoter (weak) was integrated into the genome of Pichia pastoris HEME-3 strain P TEF1UP-g1 site, strains HEME-4 and HEME-5 were produced respectively. Single colonies of Pichia pastoris recombinant strains HEME-3, HEME-4 and HEME-5 were inoculated into 50 mL culture tubes containing 5 mL YPD liquid medium (10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose) and cultured overnight at 30 ° C and 250 rpm. Subsequently, 0.1 mL of the overnight culture was inoculated into a 250 mL shake flask containing 50 mL YPD medium (and added with a final concentration of 150 mg / L ALA) and fermented at 30 ° C and 250 rpm for 48 hours. Under the condition of adding a final concentration of 150 mg / L ALA to the fermentation medium, the heme accumulation of HEME-4 and HEME-5 strains was enhanced by 86.99% and 90.24%, respectively, compared with the control HEME-3 strain (Figure 7a). The results showed that the heme accumulation of HEME-4 and HEME-5 strains increased by 86.99% and 90.24%, respectively, compared with the control HEME-3 strain (Figure 7a). GAP and P G7 Both synthetic protein scaffolds driven by the HEME-5 strain significantly promoted heme synthesis. Therefore, the HEME-5 strain was selected for subsequent experiments.

[0097] Example 9 Alleviating the accumulation of intermediate coproporphyrinogen III (CPG III) and inhibiting heme degradation

[0098] The strategy of integrating HEME-1, HEME-2 and HEME-5 strains was to express a single HEM1 gene cassette (P GAP -HEM1) was integrated into the AOXTTDOWN site of the Pichia pastoris HEME-5 genome and the wild-type HEM14 and HEM15 genes on the HEME-5 genome were replaced by their MLS truncated versions HEM14. 71-561 and HEM15 71-375 Then, Pichia pastoris genomic DNA and pGAPZαA plasmid were used as templates to amplify P HEM13 The upstream and downstream homology arms (1kb) and P GAP The promoter was constructed by overlapping extension PCR. GAP The natural promoter of the HEM13 gene on the genome of the Pichia pastoris HEME-6 strain (P HEM13 ) is replaced by P GAPThe HEME-7 strain was generated by using a promoter. Next, the upstream and downstream homology arms (1 kb) of the heme oxygenase HMX1 gene were amplified using the Pichia pastoris genome as a template. These fragments were then linked using overlap extension PCR to construct a knockout cassette for the HMX1 gene. CRISPR / Cas9 technology was then used to knock out the HMX1 gene (Gene ID: 8199419) in the HEME-5 genome of Pichia pastoris, generating the HEME-8 strain.

[0099] Single colonies of Pichia pastoris recombinant strains HEME-6, HEME-7, and HEME-8 were inoculated into 50 mL culture tubes containing 5 mL YPD liquid medium (10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose) and cultured overnight at 30°C and 250 rpm. Subsequently, 0.1 mL of the overnight culture was inoculated into a 250 mL shake flask containing 50 mL YPD medium and fermented at 30°C and 250 rpm for 48 hours. The fermentation results showed that the heme titers in HEME-7 and HEME-8 strains were 1.83 times and 2.66 times that of the HEME-6 strain, respectively (Figure 7b). This indicates that increased expression of Hem13p (shown in SEQ ID NO.16) and the deletion of the HMX1 gene (shown in SEQ ID NO.28) can greatly improve the supply of intracellular heme. Subsequently, HMX1 was knocked out in the HEME-7 strain to generate the final heme-supplying strain HEME-9. The HEME-9 strain achieved the highest heme production of 2.93 ± 0.27 mg / L (Figure 7c), which was 37.58-fold higher than that of the original strain X33-Δku70.

[0100] Example 10 Analysis of the ability of recombinant strains to biosynthesize heme proteins

[0101] On the basis of the strain HEME-9 constructed in Example 9, the strategy of constructing the P1 strain in Example 5 (replacing P GAP -Mit1 integration in P AOX1 The UP-g2 site and the Yps1-1 protease knockout were applied to the strain to produce the final production strain P1H9. Then, the porcine myoglobin (P-Mb) shown in SEQ ID NO.31, the soy leghemoglobin (S-Hb) shown in SEQ ID NO.33, the Vitreoscilla hemoglobin (V-Hb) shown in SEQ ID NO.37 and the cytochrome P450-BM3 mutant (BM3) shown in SEQ ID NO.38 were synthesized using the integration expression plasmid pPICZαA. mut) genes were integrated into the Pichia pastoris strain P1H9, and the recombinant strains P1H9-(P-Mb), P1H9-(S-Hb), P1H9-(V-Hb) and P1H9-BM3 were obtained. mut The single colonies of these recombinant Pichia pastoris strains were inoculated into 250 mL shake flasks containing 50 mL YPD medium (10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose) and grown at 30°C with shaking at 250 rpm until the logarithmic phase (OD 600 =2-6). Next, the seed culture medium was centrifuged at 3000×g for 5 minutes, and the supernatant was discarded. Subsequently, the cell pellet was resuspended twice with physiological saline to remove residual sugar. Finally, the treated cells were transferred to a 250 mL shake flask, and 50 mL of BMMY medium (1% (v / v) methanol, 10 g / L yeast powder, 20 g / L peptone, 13.4 g / L YNB, 0.4 mg / L biotin, 100 mM potassium phosphate buffer, pH 6.0) was added, and shaken at 250 rpm at 30°C; methanol was supplemented every 24 hours to a final concentration of 1%. The results of fermentation for 48 hours showed that due to sufficient heme supply in the cells, the synthesis of various heme proteins in the P1H9 strain was significantly enhanced. At the shake flask level, porcine myoglobin (P-Mb), soybean hemoglobin (S-Hb), and Vitreous Oscillator hemoglobin (V-Hb) were significantly enhanced compared with P450-BM3. mut The yields of -1 , 286.53±14.29mg L -1 、66.30±7.05mg L -1 and 57.27±6.79mg L -1 (Figure 8a). Due to the wide application of these heme proteins in the fields of artificial meat substitutes, high cell density fermentation and biocatalysis, it is very necessary to thoroughly study their biochemical properties. First, the absorption spectra of these heme proteins were detected between 350-700nm (Figure 8b). It was found that P-Mb, S-Hb and V-Hb showed a strong Soret absorption band at 414nm, that is, the heme bound to the globin was the cause of this characteristic spectrum. However, compared with the wild-type P450-BM3 (λ = 418nm), P450-BM3 mut The Soret band of α-glucose ...

[0102] Example 11 Analysis of the heme binding rate of heme-like proteins synthesized by recombinant strains

[0103] The heme binding rate of the synthesized heme protein in the strain P1 constructed in Example 5 and the strain P1H9 constructed in this example was analyzed (Figure 8c). The UV-visible absorption spectrum of the purified heme protein at 350-700 nm was recorded using a microplate reader (BioTek Synergy H1). The correct binding of heme in the heme protein was studied using the difference spectrum between the reduced and oxidized samples. 100 μL of purified heme protein was mixed with 100 μL of solution I (40% (v / v) pyridine, 0.2 M NaOH and 500 μM potassium ferricyanide) in a 96-well microplate. The mixture was scanned (500-600 nm) to obtain the oxidation spectrum. Then, 2 μL of solution II (0.5 M sodium dithionite in 0.5 M NaOH) was added to the oxidized sample. The mixture was scanned (500-600 nm) to obtain the reduction spectrum. Quantification was performed by Beer-Lambert quantification using the extinction coefficient of pyridine hemochromagen of 23.98 mM. -1 cm -1 The heme content in heme proteins was calculated. The heme binding rate was expressed as ([mol of heme] / [mol of globin])×100%. The results showed that from strains P1 to P1H9, P-Mb, S-Hb, V-Hb and P450-BM3 mut The heme binding rates of V-Hb increased by 2.87 times, 3.67 times, 1.07 times and 2.04 times, reaching 60.02±6.98%, 79.47±6.72%, 25.94±4.36% and 61.27±6.86%, respectively. Among them, the increase in the heme binding rate of V-Hb was not very obvious, which may be related to the low assembly efficiency of apo-(V-Hb) and heme at 30°C.

[0104] Example 12 Analysis of the functional activity of heme-like proteins synthesized by recombinant strains

[0105] (1) Investigate the relative peroxidase activity of P-Mb and S-Hb:

[0106] The specific peroxidase activity of purified P-Mb and S-Hb samples was determined using 3,3′,5,5′-Tetramethylbenzidine (TMB) Chromogen Solution (P0209, Beyotime). As shown in Figure 8d, the specific peroxidase activity of P-Mb and S-Hb increased by 38.10% and 29.31% from strains P1 to P1H9, respectively, reaching 691.13 ± 13.37 U mg -1 and 429.35±9.42 U mg -1 .

[0107] (2) Evaluation of the oxygen binding properties of V-Hb:

[0108] Sodium dithionite (SDT) was used as an oxygen scavenger and reducing agent for V-Hb. In a 96-well microplate, 100 μL of sample was mixed with 10 μL of SDT solution (10 mg / mL). After the SDT was consumed, deoxy V-Hb was gradually converted to oxidized V-Hb. As oxidized V-Hb was formed, the absorbance at 556 nm (A 556 ) drops sharply and at 523nm(A 523 ) is observed at an isosbestic point. The oxygen binding assay for V-Hb is reported as Sat. O2 (oxygen saturation) time course.

[0109] where K Hb is the absorbance ratio of completely deoxygenated V-Hb; K HbO2 is the absorbance ratio of fully oxygenated V-Hb; K x A is the absorbance ratio at a specific time after the start of oxygenation. A microplate reader (BioTek Synergy H1) was used to record A 556 and A 523 As shown in Figure 8e, the transition from a deoxygenated to a fully oxygenated state occurred faster in V-Hb biosynthesized by the P1H9 strain (12 min, 91.16% oxygenation) than in V-Hb synthesized by the P1 strain (22 min, 90.22% oxygenation), indicating that the oxygen-binding capacity of V-Hb was enhanced.

[0110] (3) Analysis of P450-BM3 expressed in strains P1 and P1H9 mut Catalytic efficiency for the conversion of phenol to hydroquinone:

[0111] Will express BM3 mut2mL fermentation broth of the recombinant Pichia pastoris strain was centrifuged at 6000rpm for 5 minutes (the cell amount was basically the same). The cell pellet was collected and resuspended in 4mL of potassium dihydrogen phosphate buffer (100mM, pH 8.0) containing 0.05g / mL glucose and 10mM substrate phenol. The reaction was carried out in a 50mL culture tube and reacted at 250rpm for 8h. However, 100μL of the reaction solution was mixed with 900μL of methanol and centrifuged at 12000rpm for 5 minutes. Then, the supernatant was separated and analyzed by HPLC. The conversion rate is expressed as ([mM product] / [10mM substrate])×100%. HPLC analysis: flow rate 0.5mL / min, methanol containing 0.1% TFA (trifluoroacetic acid) and water ratio of 45 / 55, column temperature 35℃, injection volume 10μL, detection wavelength 220nm. The results showed that the whole cell P450-BM3 of the P1H9 strain mut The catalytic efficiency was 8.47 times that of the P1 strain, reaching 29.56 ± 0.33% (Figure 8f). Therefore, these results indicate that improving the intracellular heme supply is beneficial for the production of highly active heme proteins.

[0112] Example 13 Expression of bovine myoglobin, clover hemoglobin, and porcine hemoglobin in a heme-supplying Pichia pastoris chassis

[0113] Finally, to provide more myoglobin and hemoglobin from animal and plant sources for the development of artificial meat, the integrative expression plasmid pPICZαA was used to attempt secretory expression of bovine myoglobin (B-Mb, shown in SEQ ID NO.32), clover hemoglobin (C-Hb, shown in SEQ ID NO.34), porcine hemoglobin α subunit (P-Hbα, shown in SEQ ID NO.35), and porcine hemoglobin β subunit (P-Hbβ, shown in SEQ ID NO.36) in the heme-supplying Pichia pastoris chassis strain P1H9. The results showed that the P1H9 strain was also suitable for expressing B-Mb, C-Hb, P-Hbα, and P-Hbβ, with yields of 20.04±3.21 mg L, respectively. -1 , 5.02±1.68mg L -1 , 10.16±4.62mg L -1 and 5.24±1.24 mg L -1 , the heme binding rates were 40.15±5.32%, 15.46±2.16%, 18.27±2.31% and 13.24±2.05%, respectively ( FIG9 ).

[0114] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered bacterium, characterized in that: At least one of the following improvements was made based on the starting strain: (1) Knockout or silence gene ku70; (2) integrating a transcription activator or a molecular chaperone into the genome of the starting strain; the transcription activator includes Mit1, Mxr1 or Prm1, and the molecular chaperone includes PDI or KAR2; (3) knocking out the protease gene responsible for degrading globin in the genome of the starting strain; the protease includes Pep4, Prb1 or Yps1-1; (4) Knockout the mitochondrial localization signal peptides of Hem1p, Hem14p, and Hem15p in the Pichia pastoris genome; (5) Introducing genes for the C5 synthesis pathway; the genes encoding the C5 synthesis pathway are derived from Escherichia coli and Bacillus subtilis; (6) fixing the rate-limiting enzyme Hem2p, Hem3p or Hem4p fused with a specific ligand to a protein scaffold containing a metazoan signal protein interaction domain for spatial assembly and integrating it into the genome; the protein scaffold has a structure shown as GBD domain-linker-SH3 domain-linker-PDZ domain; (7) Use P GAP The promoter enhances the expression of the HEM13 gene; (8) Knockout or silence the heme oxygenase HMX1 gene.

2. The genetically engineered bacterium according to claim 1, characterized in that: The transcription activator or the molecular chaperone is integrated into the genome AOX1 UP-g2 site.

3. The genetically engineered bacterium according to claim 1 or 2, characterized in that: The protein scaffold GBD domain-linker-SH3 domain-linker-PDZ domain is integrated into the genome TEF1 UP-g1 site, and by the promoter P GAP or promoter P G7 Regulate expression.

4. The genetically engineered bacterium according to any one of claims 1 to 3, characterized in that: The genes HEM1 and C5 biosynthesis pathways are expressed through promoter P GAP or P AOX1 Regulate expression.

5. The genetically engineered bacterium according to any one of claims 1 to 4, characterized in that: The genetically engineered bacteria also express heme proteins; the heme proteins include one or more of porcine myoglobin P-Mb, bovine myoglobin B-Mb, soy hemoglobin S-Hb, clover hemoglobin C-Hb, porcine hemoglobin P-Hb, Vitreoscilla hemoglobin V-Hb, and wild-type cytochrome P450-BM3 mutants.

6. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The genetic engineering bacteria uses Pichia pastoris or Saccharomyces uvarum as a host.

7. The genetically engineered bacterium according to claim 6, characterized in that: Using Pichia pastoris X33 as the starting strain, the gene is expressed by integrating the expression plasmid, or the gene is integrated into the Pichia pastoris genome through CRISPR / Cas9 technology for expression.

8. A method for producing heme and / or heme-like proteins by fermentation, characterized in that: The genetically engineered bacteria according to any one of claims 1 to 6 are used as fermentation microorganisms, and fermented at 25 to 35° C. in a fermentation medium for at least 24 hours.

9. The method according to claim 8, characterized in that The fermentation medium includes, but is not limited to, YNB, YPD, BMGY or BMMY medium.

10. Use of the genetically engineered bacteria according to any one of claims 1 to 7, or the method according to any one of claims 8 to 9 in producing products in the fields of food, biology or medicine.

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