A method for improving heme synthesis in recombinant Escherichia coli

Abstract

The invention discloses a method for improving the synthesis of recombinant Escherichia coli heme, and belongs to the field of genetic engineering. The present invention solves the problem of key precursor excretion by knocking out the protein gene encoding the excretion of precursors, causing PPIX accumulation, and further improves the recombinant Escherichia coli heme synthesis ability by optimizing the iron ion concentration, glutamyl-tRNA reductase (HemA), the expression of a protein involved in iron transport (EfeB) and ferrochelatase (HemH). Compared with the starting strain, the growth condition does not change significantly, but the intracellular heme content is greatly improved, and the heme concentration is about 4 times that of the starting strain, which has good application prospects.

Description

Technical Field

[0001] The invention relates to a method for improving the synthesis of recombinant Escherichia coli heme, and belongs to the field of genetic engineering. Background Art

[0002] Heme is a common metalloporphyrin, which is formed by chelating an Fe in the center of protoporphyrin IX (PPIX) through ferrochelatase. 2+ The product formed is also called iron porphyrin, which can be used as a colorant and iron supplement in food processing and manufacturing. It can also be used as a broad-spectrum dark quencher for fluorescent detection of protein function under pathological conditions and has a wide range of applications. The current chemical synthesis and the use of organic extraction or enzymatic hydrolysis of biomaterials to prepare heme are complex, low-yielding, time-consuming, and environmentally unfriendly. The use of recombinant bacteria to biosynthesize heme is the most promising method for large-scale production of heme, but the current yield is low and not suitable for industrial production. Therefore, it is very necessary to improve the heme synthesis capacity of recombinant bacteria through metabolic engineering.

[0003] Escherichia coli (E. coli), a key host for genetic engineering, primarily synthesizes heme via the C5 pathway using glutamate as a precursor. Enhancing key enzyme genes in this metabolic pathway can increase the production of key precursors and has therefore been used to boost heme content. For example, overexpressing hemA, which encodes glutamyl t-RNA reductase, can enhance the synthesis of the precursor 5-aminolevulinic acid (5-ALA). However, this also promotes 5-ALA excretion from the cell, wasting cellular resources. Therefore, if metabolic engineering can address the problem of precursor excretion and further redirect metabolic flux toward heme, it is expected to enhance the cell's heme biosynthesis capacity. Summary of the Invention

[0004] The present invention solves the problem of key precursor excretion by knocking out the protein gene encoding the excretion of the precursor, resulting in PPIX accumulation. The heme synthesis capacity of the recombinant Escherichia coli is further improved by optimizing the iron ion concentration, the expression of glutamyl-tRNA reductase (HemA), the protein involved in iron transport (EfeB), and the ferrochelatase (HemH).

[0005] The present invention provides a genetically engineered bacterium, wherein the coding genes of the outer membrane channel protein TolC and / or the transport protein RhtA are knocked out, and / or at least one of the glutamyl-tRNA reductase HemA, the protein EfeB involved in iron transport, or the ferrochelatase HemH is overexpressed.

[0006] In one embodiment, the genetically engineered bacteria knock out the genes encoding the outer membrane channel protein TolC and the transport protein RhtA;

[0007] Alternatively, the genetically engineered bacteria knock out the gene encoding the outer membrane channel protein TolC and overexpress glutamyl-tRNA reductase HemA and ferrochelatase HemH;

[0008] Alternatively, the genetically engineered bacteria have knocked out the genes encoding the outer membrane channel protein TolC and the transport protein RhtA, and overexpressed the protein EfeB and the ferrochelatase HemH involved in iron transport;

[0009] Alternatively, the genetically engineered bacteria knock out the genes encoding the outer membrane channel protein TolC and the transport protein RhtA, and overexpress glutamyl-tRNA reductase HemA and ferrochelatase HemH.

[0010] In one embodiment, the nucleotide sequence of the gene tolC encoding the outer membrane channel protein TolC is shown as SEQ ID NO.1, the nucleotide sequence of the gene hemA encoding the glutamyl-tRNA reductase HemA is shown as SEQ ID NO.2, the nucleotide sequence of the gene hemH encoding the ferrochelatase HemH is shown as SEQ ID NO.3, the nucleotide sequence of the gene rhtA encoding the transport protein RhtA is shown as SEQ ID NO.4, and the nucleotide sequence of the gene efeB encoding the protein EfeB involved in iron transport is shown as SEQ ID NO.5.

[0011] In one embodiment, the overexpression is to connect the gene to a pET vector for episomal expression.

[0012] In one embodiment, the pET vector is pET28a.

[0013] In one embodiment, the genetically engineered bacteria uses Escherichia coli BL21 (DE3) as a starting strain.

[0014] The present invention provides a method for improving the heme synthesis ability of Escherichia coli, which comprises knocking out the coding genes of the outer membrane channel protein TolC and / or the transport protein RhtA on the starting bacterial genome, and / or overexpressing at least one of the glutamyl-tRNA reductase HemA, the protein EfeB involved in iron transport, or the ferrochelatase HemH.

[0015] The present invention provides a method for producing hemoglobin, wherein the genetically engineered bacteria are used as fermentation strains, and the 2+ and / or Fe 3+ Hemoglobin is produced by fermentation in a culture medium.

[0016] In one embodiment, the genetically engineered bacteria are activated overnight and inoculated into a culture medium containing Fe at a volume ratio of 1 to 5%. 2+ and / or Fe3+ in the culture medium.

[0017] In one embodiment, the culture medium contains Fe 2+ Concentration ranges from 0 to 200 μmol·L -1 , Fe 3+ Concentration ranges from 0 to 50 μmol·L -1 .

[0018] In one embodiment, the culture is carried out at 30-37° C. for 8-16 hours.

[0019] The present invention also protects the use of the genetically engineered bacteria or the method in producing heme and / or products containing heme.

[0020] Beneficial effects:

[0021] (1) The present invention knocks out the gene tolC involved in encoding PPIX excretion in the Escherichia coli genome by Red homologous recombination, overexpresses hemA and hemH, and exogenously adds Fe 2+ , promoting the conversion of PPIX to heme, and the heme content of the strain reached 34.69μmol / L after 12h of fermentation production; optimizing Fe 2+ The added amount can eventually achieve 40.18μmol / L hemoglobin.

[0022] (2) The present invention knocks out the genes rhtA and tolC involved in encoding 5-ALA and PPIX excretion in the Escherichia coli genome by Red homologous recombination, and exogenously adds Fe 2+ and Fe 3+ , promoting the conversion of PPIX to heme, and exogenously added Fe 2+ The concentration is 100 μmol·L -1 When the heme content reached the highest value of 10.12 μmol / L after 12 h of fermentation, the heme content reached the highest value of 10.12 μmol / L. -1 Fe 3+ When the fermentation is carried out for 12 hours, the hemoglobin concentration can reach 12.77 μmol / L.

[0023] (3) The present invention knocks out the genes rhtA and tolC involved in encoding 5-ALA and PPIX excretion in the Escherichia coli genome by Red homologous recombination, overexpresses efeB and hemH, and exogenously adds Fe 2+ , promoting the conversion of PPIX to hemoglobin, and the hemoglobin content after 12 hours of fermentation production was 10μmol / L.

[0024] (4) The present invention knocks out the genes rhtA and tolC involved in encoding 5-ALA and PPIX excretion in the Escherichia coli genome by Red homologous recombination, overexpresses hemA and hemH, and exogenously adds Fe 2+ and Fe 3+ , promoting the conversion of PPIX to hemoglobin, and the hemoglobin content after 12 hours of fermentation production was 42.1μmol / L.

[0025] (5) The growth condition of the genetically engineered bacteria constructed by the present invention did not change significantly compared with the starting strain, but the intracellular hemoglobin content was greatly increased, and the hemoglobin concentration was about 4 times that of the starting strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 2 shows the heme content and OD of each strain. 600 .

[0027] Figure 2 The growth curves of each strain are shown. DETAILED DESCRIPTION

[0028] 1. LB liquid medium (g / L): tryptone 10%, yeast powder 5%, NaCl 10%, pH 7.0.

[0029] 2. Hemoglobin concentration determination:

[0030] The hemoglobin concentration was detected by fluorescence method, specifically: an appropriate amount of cultured cells was taken to make the OD 600 × bacterial liquid volume (mL) = 8 (for example, if the OD 600 value is 0.4, then take 20 mL of bacterial liquid), centrifuge at 4°C, 12000 r / min for 5 min to obtain a bacterial precipitate, wash with water and add it to a 1.5 mL amber centrifuge tube; add 500 μL of 20 mmol / L oxalic acid to the 1.5 mL centrifuge tube, and let it stand in a dark room at 4°C overnight for 16 h; add 500 μL of 2 mol / L oxalic acid to each centrifuge tube, take half of the sample (for porphyrin concentration detection) and react at room temperature as the experimental control group, and the other half of the sample (for porphyrin and hemoglobin total concentration detection) is heated at 95°C for 30 min; after natural cooling, centrifuge at 12000 r / min for 5 min, take 200 μL and place it in a black 96-well plate for fluorescence value detection (excitation wavelength is 400 nm, emission wavelength is 620 nm), and the difference between the two groups is the hemoglobin concentration to be measured.

[0031] 3. Preparation of competent E. coli: Refer to the instructions of Takara Competent Cell Preparation Kit.

[0032] Example 1

[0033] Based on the nucleotide sequence of the tolC gene as shown in SEQ ID NO.1, 500 bp upstream and downstream homology arms were designed, and the tolC gene on the wild-type Escherichia coli BL21 (DE3) (WT) genome was knocked out by Red homologous recombination (for specific steps, see Datsenko KA. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(12): 6640-6645). After sequencing verification, a genetically engineered bacterium with the tolC gene knocked out was obtained and named WT-ΔT.

[0034] The hemA gene with a nucleotide sequence as shown in SEQ ID NO.2 and the hemH gene with a nucleotide sequence as shown in SEQ ID NO.3 were respectively ligated between the EcoRI, Hind III, and Xho I restriction sites of the vector pET28a to construct the plasmid pEAH, and then introduced into WT-ΔT competent cells to obtain the strain WT-ΔT-AH.

[0035] The wild-type Escherichia coli BL21 (DE3), WT-ΔT and WT-ΔT-AH strains were inoculated into shake flasks containing 50 mL of LB liquid medium, respectively. After overnight activation in a shaker at 37°C and 200 r / min, they were inoculated into 150 mL of LB liquid medium at a 2% (v / v) inoculum size and cultured for 12 h. The plasmid-containing strain WT-ΔT-AH was added with Kan antibiotics during the culture process and IPTG with a final concentration of 0.2 mmol / L was added for induction for 10 h after culturing for 2 h. The heme content was detected. The heme content of the WT-ΔT strain reached 8.35 μmol / L, and the heme content of the WT-ΔT-AH strain reached 34.69 μmol / L.

[0036] Example 2

[0037] FeSO4 was added to LB liquid culture medium at final concentrations of 0, 40, 80, 160, and 240 μmol / L. The strain WT-ΔT-AH constructed in Example 1 was used as the heme-producing strain and was inoculated into 50 mL of LB liquid culture medium containing FeSO4. After overnight activation in a shaker at 37°C and 200 r / min, the strain was inoculated into 150 mL of LB liquid culture medium containing FeSO4 at an inoculum size of 2% (v / v) and cultured for 12 h. Kan antibiotics were added and after culturing for 2 h, IPTG was added at a final concentration of 0.2 mmol / L to induce for 10 h.

[0038] The results are as follows Figure 1 As shown, the heme concentration was the highest when 80 μmol / L FeSO4 was added, reaching 40.18 μmol / L.

[0039] Example 3

[0040] The rhtA gene with the nucleotide sequence shown in SEQ ID NO.4 on the genome of wild-type Escherichia coli BL21 was knocked out using the same method as in Example 1, and the tolC gene with the nucleotide sequence shown in SEQ ID NO.1 was further knocked out to obtain strain WT-RT.

[0041] The strains WT-RT and WT were inoculated into shake flasks containing 50 mL of LB liquid medium. After overnight activation in a shaker at 37°C and 200 rpm, the strains were inoculated into 150 mL of LB liquid medium at a 2% (v / v) inoculum and cultured for 12 hours. The bacterial concentration was measured in real time, and growth curves were generated. There was no significant difference in the growth of the two strains compared to the WT ( Figure 2 ).

[0042] 0, 25, 50, 75, and 100 μmol / L Fe were added to LB medium. 2+ and Fe 3+ , when exogenously added Fe 2+ The concentration is 100 μmol·L -1 When 25 μmol·L -1 Fe 3+ When WT-RT was treated with RT-glucose 5-nitropropene, the hemoglobin concentration of WT-RT could reach 12.77 μmol / L.

[0043] Example 4

[0044] The efeB gene with the nucleotide sequence shown in SEQ ID NO.5 and the hemH gene with the nucleotide sequence shown in SEQ ID NO.3 were ligated between the EcoR I and Xho I restriction sites of the vector pET28a to construct the plasmid pEEH, and the WT-RT constructed in Example 3 was introduced to obtain the strain RT-EHE.

[0045] The strain RT-EHE was inoculated into 50 mL LB liquid medium (containing a final concentration of 150 μmol·L -1 Fe 2+ ) in a shaker at 37°C and 200 r / min overnight, and then inoculated into 150 mL of LB liquid medium (containing a final concentration of 150 μmol·L -1 Fe 2+ ) for 12 h, and its hemoglobin content was 10 μmol / L.

[0046] Example 5

[0047] The plasmid pEAH obtained in Example 1 was introduced into the strain WT-RT constructed in Example 3 to obtain the strain WT-RT-pEAH. The strains WT-RT-pEAH and WT were inoculated in 50 mL LB liquid medium (containing a final concentration of 150 μmol·L -1 Fe 2+ ) for 12 h, and its hemoglobin content was 42.1 μmol·L -1 Compared with WT, the heme concentration of WT-RT-pEAH was 4 times higher.

[0048] 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: The genetically engineered bacteria is based on Escherichia coli as a starting strain, wherein the gene encoding the outer membrane channel protein TolC is knocked out and glutamyl-tRNA reductase HemA and ferrochelatase HemH are overexpressed; the gene encoding the glutamyl-tRNA reductase HemA is hemA The nucleotide sequence of the gene encoding the ferrochelatase HemH is shown in SEQ ID NO.

2. hemH The nucleotide sequence is shown in SEQ ID NO.

3.

2. A genetically engineered bacterium according to claim 1, characterized in that: The genetically engineered bacteria also knocks out the gene encoding the transport protein RhtA.

3. The genetically engineered bacterium according to claim 1 or 2, characterized in that Gene encoding the outer membrane channel protein TolC tolC The nucleotide sequence of the transporter protein RhtA is shown in SEQ ID NO.

1. rhtA The nucleotide sequence is shown in SEQ ID NO.

4.

4. The genetically engineered bacterium according to claim 1, characterized in that The overexpression is to connect the gene to the pET vector for free expression.

5. The genetically engineered bacterium according to claim 3, characterized in that The overexpression is to connect the gene to the pET vector for free expression.

6. The genetically engineered bacterium according to claim 1 or 2, characterized in that Escherichia coli BL21 (DE3) was used as the starting strain.

7. The genetically engineered bacterium according to claim 3, characterized in that Escherichia coli BL21 (DE3) was used as the starting strain.

8. A method for improving the heme synthesis ability of Escherichia coli, characterized in that: The method comprises knocking out the genes encoding the outer membrane channel protein TolC and the transport protein RhtA on the genome of the starting bacteria, overexpressing the glutamyl-tRNA reductase HemA and the ferrochelatase HemH; the gene encoding the glutamyl-tRNA reductase HemA hemA The nucleotide sequence of the gene encoding the ferrochelatase HemH is shown in SEQ ID NO.

2. hemH The nucleotide sequence is shown in SEQ ID NO.

3.

9. A method for producing hemoglobin, characterized in that The genetically engineered bacteria according to any one of claims 1 to 7 are used as fermentation strains, in a culture containing Fe 2+ and / or Fe 3+ Hemoglobin is produced by fermentation in a culture medium.

10. The method according to claim 9, characterized in that After overnight activation, the genetically engineered bacteria according to any one of claims 1 to 7 are inoculated into a culture medium containing Fe at a volume ratio of 1 to 5%. 2+ and / or Fe 3+ in the culture medium.

11. The method according to claim 9 or 10, characterized in that The Fe 2+ Concentration ranges from 40 to 200 μmol·L -1 , Fe 3+ The concentration is 25-50 μmol·L -1 .

12. Use of the genetically engineered bacteria according to any one of claims 1 to 7, or the method according to any one of claims 9 to 11, in the production of heme and / or products containing heme.

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