Method for improving heme synthesis of genetically engineered escherichia coli and application thereof

Abstract

This invention discloses a method for enhancing heme synthesis in genetically engineered *Escherichia coli* and its application, belonging to the field of genetic engineering technology. This invention involves knocking out the endogenous GluTR encoding gene in *E. coli* BL21(DE3). hemA We obtained strain WTΔA and blocked the C5 pathway; then we used Rhodopseudomonas aeruginosa... Rs ALAS genes hemA R Import WTΔA to construct a strain containing only the C4 pathway, WTΔA-pC / A. R ; further Rs The cysteine ​​at position 398 of ALAS was mutated to alanine to obtain strain WTΔA-pC / C398A, which increased the yield of 5-ALA. Finally, ferrous chelate was further expressed on the basis of WTΔA-pC / C398A to obtain recombinant strain WTΔA-pC / C398AH, which increased the yield of heme to 209.63 μmol / L.

Description

Technical Field

[0001] This invention relates to a method for improving the synthesis of heme in genetically engineered Escherichia coli and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] Products of the heme biosynthetic pathway, such as 5-aminolevulinic acid (5-ALA), protoporphyrin, and heme, have significant applications in food processing, photodynamic therapy, iron supplementation, and other food and medical fields. Chemical synthesis and extraction of heme and related metabolites from biological materials are complex, low-yield, time-consuming, and environmentally unfriendly. Recombinant microbial synthesis is a promising method for obtaining these products, but current yields are low and unsuitable for industrial production.

[0003] Heme synthesis in organisms can be divided into two pathways, C4 and C5, based on the synthesis of the precursor 5-ALA. In the C4 pathway, 5-ALA synthase (ALAS) catalyzes the condensation of L-glycine and succinyl-CoA to form 5-ALA, requiring only one step. In the C5 pathway, glutamate-tRNA synthase catalyzes the formation of glutamyl-tRNA from L-glutamate, which is then catalyzed by glutamyl-tRNA reductase to form glutamate-1-semialdehyde. The latter is then catalyzed by glutamyl-1-semialdehyde-2,1-mutase to generate 5-ALA, requiring three steps.

[0004] Escherichia coli ( Escherichia coli As an important genetically engineered host bacterium, E. coli possesses a clear genetic background and is easy to culture, making it one of the key chassis strains for heme production. It synthesizes 5-ALA via the C5 pathway. After 5-ALA undergoes multiple enzymatic reactions to generate protoporphyrin IX (PPIX), and finally, ferrous chelase chelates ferrous ions in PPIX to form heme. Existing literature mainly focuses on enhancing intracellular heme synthesis based on the C5 pathway by strengthening the expression of key enzymes in 5-ALA synthesis; or dividing the metabolic pathway into different modules and overexpressing key enzyme genes to regulate the expression intensity of each module; or reducing byproduct generation through gene knockout. However, reports on structural modifications of key enzymes in the metabolic pathway are very limited. Introducing the C4 pathway into E. coli is another way to enhance heme synthesis metabolism, but the coexistence of the original C5 pathway leads to insufficient supply of precursors such as succinyl-CoA.

[0005] Therefore, improving the ability of engineered bacteria to synthesize heme metabolites through metabolic engineering and enzyme engineering is a key bottleneck in achieving high-yield heme production through microbial fermentation. Summary of the Invention

[0006] To address the aforementioned problems, this invention knocks out the gene encoding endogenous GluTR in Escherichia coli BL21(DE3). hemA ) obtained strain WTΔA, blocking the C5 pathway; then from Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroides )of Rs The gene encoding ALAS ( hemA R Import WTΔA to construct a strain containing only the C4 pathway, WTΔA-pC / A. R ; further Rs The cysteine ​​at position 398 of ALAS was mutated to alanine to obtain strain WTΔA-pC / C398A, which increased the yield of 5-ALA. Finally, ferrous chelate was further expressed on the basis of WTΔA-pC / C398A to obtain recombinant strain WTΔA-pC / C398AH, which increased the yield of heme to 209.63 μmol / L.

[0007] The first objective of this invention is to provide a recombinant *E. coli* strain that synthesizes heme, wherein the gene encoding aminoacyl-tRNA reductase (GluTR) is knocked out and overexpressed. Rs ALAS or its mutants; the Rs The amino acid sequence of ALAS is shown in SEQ ID NO: 2; the mutant is the one with the amino acid sequence shown in SEQ ID NO: 2. Rs The ALAS was obtained by mutating cysteine ​​at position 398 to alanine.

[0008] In one embodiment, the *E. coli* simultaneously overexpresses ferrous chelate; the amino acid sequence of the ferrous chelate is shown in SEQ ID NO: 6.

[0009] In one embodiment, the amino acid sequence of the aminoacyl-tRNA reductase (GluTR) is shown in SEQ ID NO: 1; In one embodiment, the recombinant Escherichia coli is constructed based on Escherichia coli BL21(DE3).

[0010] A second objective of this invention is to provide a 5-aminolevulinic acid synthase mutant, wherein the mutant has an amino acid sequence as shown in SEQ ID NO: 2. Rs The ALAS was obtained by mutating cysteine ​​at position 398 to alanine.

[0011] A third objective of this invention is to provide a gene encoding the aforementioned 5-aminolevulinic acid synthase mutant or a recombinant vector carrying the aforementioned gene.

[0012] A fourth objective of this invention is to provide a method for increasing heme production in *Escherichia coli*, wherein the gene encoding aminoacyl-tRNA reductase (GluTR) is knocked out in *E. coli*, and ferrous chelate is overexpressed. Rs ALAS or its mutants; The amino acid sequence of the aminoacyl-tRNA reductase (GluTR) is shown in SEQ ID NO: 1; Rs The amino acid sequence of ALAS is shown in SEQ ID NO: 2; the mutant is the one with the amino acid sequence shown in SEQ ID NO: 2. Rs The ALAS was obtained by mutating cysteine ​​at position 398 to alanine; the amino acid sequence of the ferrous chelate is shown in SEQ ID NO: 6.

[0013] A fifth object of the present invention is to provide a method for producing heme, the method comprising fermentation production using any of the recombinant Escherichia coli described above.

[0014] In one embodiment, the fermentation involves inoculating recombinant Escherichia coli into a fermentation medium at an inoculum size of 1-5% v / v and culturing at 30-37°C until OD reaches 100%. 600 When the concentration is 0.6~0.8, add IPTG to a final concentration of 0.1~0.5 mmol / L and induce at 16~37℃ for 10~24 h.

[0015] In one embodiment, the fermentation medium may also contain L-Gly at a concentration of 50-400 mg / L.

[0016] A sixth object of the present invention is to provide the use of any of the above-described recombinant Escherichia coli or the method described above in the synthesis of heme or in the preparation of products containing heme.

[0017] Beneficial effects This invention uses Escherichia coli BL21(DE3) as the expression host and knocks out the gene encoding GluTR. hemA ), constructed strain WTΔA, to block the endogenous C5 pathway in Escherichia coli; simultaneously, from Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroides )of Rs ALAS genes hemA R When the pCDFduet-1 plasmid is ligated between the Bam HI and Not I sites and transformed into WTΔA, the C4 pathway-only E. coli strain ΔA-pC / A is obtained. R .

[0018] The present invention further incorporates the above-mentioned RsThe cysteine ​​(Cys) at position 398 of ALAS was mutated to alanine (Ala) to obtain strain WTΔA-pC / C398A, which achieved a 5-ALA yield of 24.54 mg / L. Finally, ferrous chelate was further expressed on the basis of WTΔA-pC / C398A to obtain recombinant strain WTΔA-pC / C398AH, which achieved a heme yield of 209.63 μmol / L.

[0019] The heme content of the strain WTΔA-pC / C398AH constructed in this invention can be further increased to 370.18 μmol / L when 200 mg / L of L-Gly is added during fermentation. Attached Figure Description

[0020] Figure 1 This diagram illustrates the heme synthesis pathway and its regulation; where L-Gly represents L-glycine; Succinyl-CoA represents succinyl-CoA; α-KG represents α-ketoglutarate; L-Glu represents L-glutamate; Glutamyl-tRNA represents glutamyl-tRNA; Glutamate-1-semialdehyde represents glutamate-1-semialdehyde; 5-ALA represents 5-aminolevulinic acid; PPIX represents protoporphyrin IX; and Heme represents heme. Rs ALAS is 5-aminolevulinic acid synthase from Rhodotorula glutinis; GluRS is gamma-glutamyl tRNA synthase; GluTR is gamma-glutamyl tRNA reductase; GSAM is glutamate-1-semialdehyde-2,1-amino mutase; Fech is ferrous chelate synthase; the symbols in parentheses below the enzymes are their corresponding coding genes; yellow highlights indicate overexpression, red crosses indicate gene knockout; yellow lines represent the C4 pathway, blue lines represent the C5 pathway, red lines represent feedback control, and purple lines represent... Rs Mutations in ALAS. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0022] Culture media involved in the examples: Luria Bertani (LB) medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, and distilled water to a final volume of 1 L.

[0023] LB solid medium is prepared by adding 2% agar powder by mass to the LB medium formula.

[0024] The preparation method for antibiotic resistance culture medium is as follows: After sterilizing LB medium, cool it to about 50~60℃, add the corresponding antibiotics as needed, and the final concentrations are as follows: kanamycin (Kan) 50 mg / L, ampicillin (Amp) 50 mg / L, chloramphenicol (Cm) 25 mg / L, and streptomycin (Str) 50 mg / L.

[0025] The measurement methods involved in the examples are as follows: 1. Determination of intracellular heme by fluorescence method The required bacterial culture volume V (mL) was calculated using the formula OD600×V=8. The required volume of bacterial culture was centrifuged at 4 ℃ and 10500×g for 5 min, and the bacterial precipitate was collected. 500 μL of 20 mmol / L oxalic acid was added to the washed bacterial cells, and the mixture was placed in a 4 ℃ refrigerator for 16 h. Then, 500 μL of 2 mol / L oxalic acid was added, and the mixture was thoroughly mixed and aliquoted into brown centrifuge tubes. One tube was heated at 100 ℃ for 30 min, and the other was incubated at room temperature. Both tubes were centrifuged at 4 ℃ and 10500×g for 5 min. The fluorescence values ​​of the samples were measured using an excitation wavelength of 400 nm and an emission wavelength of 620 nm. The fluorescence value of the heated sample represents the total amount of heme and porphyrin, while the fluorescence value of the room temperature sample represents the porphyrin content. The difference between the two values ​​represents the heme content. Chlorhexene was used as a standard.

[0026] 2. Determination method of 5-aminolevulinic acid Take 2 mL of fermentation broth, centrifuge at 12,000 × g for 2 min, collect the supernatant, add 1 mL of acetate buffer (pH 4.6) and 0.5 mL of acetylacetone, heat in boiling water for 15 min, and allow to cool naturally to room temperature. Take 2 mL of the reaction solution, add 2 mL of DMAB colorimetric reagent, and allow the colorimetric reaction to proceed for 30 min. Use deionized water as a blank control and measure the absorbance (A554) at 554 nm. Use 5-aminolevulinate as a standard.

[0027] Example 1: Knockout of the gene encoding GluTR According to the NCBI Escherichia coliThe BL21 (DE3) genome sequence was used as a template to design upstream and downstream primers for knocking out the homologous sequences encoding the GluTR gene, as well as identification primers. The Red homologous recombination method (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) was used to knock out the GluTR (amino acid sequence of GluTR is shown in SEQ ID NO: 1) gene in *E. coli* BL21 (DE3) (named WT). hemA During the knockout process, 5-aminolevulinic acid (5-ALA) at a final concentration of 6.56 mg / L was added to the culture medium to maintain the growth of the strain. Sequencing confirmed the knockout. hemA The genetically engineered strain was named WTΔA. This strain could not grow in LB medium, but its growth was restored after exogenous addition of 6.56 mg / L 5-ALA to the medium, indicating that the C5 pathway of WTΔA was blocked.

[0028] Example 2: Expression Rs ALAS or Rs ALAS mutant 1. Expression Rs ALAS or Rs ALAS mutant From Rhodophyta ( Rhodobacter sphaeroides )of Rs ALAS ( Rs The amino acid sequence of ALAS (shown in SEQ ID NO: 2) is from the gene. hemA R By linking the pCDFduet-1 plasmid between the Bam HI and Not I sites, plasmid pC / A was obtained. R Plasmid pC / A R The strain WTΔA-pC / A was obtained by transformation into WTΔA. R .

[0029] Each RsThe 264th cysteine ​​residue of the ALAS amino acid sequence was mutated to alanine (C264A); the 341st cysteine ​​residue was mutated to alanine (C341A); and the 398th cysteine ​​residue was mutated to alanine (C398A), resulting in mutants C264A, C341A, and C398A (the amino acid sequence of mutant C264A is shown in SEQ ID NO: 3, the amino acid sequence of mutant C341A is shown in SEQ ID NO: 4, and the amino acid sequence of mutant C398A is shown in SEQ ID NO: 5). The construction method is as follows: using pC / A R Plasmids were used as reverse PCR templates to target specific targets. Rs Three pairs of site-directed mutagenesis primers were designed for cysteine ​​residues 264, 341, and 398 of ALAS (primers are shown in Table 1). Reverse PCR amplification was performed using a high-fidelity enzyme. The cells were treated with Dpn I enzyme at 37ºC for 1 h, and after template removal, transformed into WTΔA for expression. Positive clones were screened, plasmids were extracted, and sequencing confirmed the results, yielding strain WTΔA-pC / A. R , WTΔA-pC / C264A, WTΔA-pC / C341A and WTΔA-pC / C398A.

[0030] Table 1 Primers

[0031] 2. Determination of 5-aminolevulinic acid (5-ALA) content strains WT, WTΔA-pC / A R WTΔA-pC / C264A, WTΔA-pC / C341A, and WTΔA-pC / C398A were inoculated into LB medium containing streptomycin at an inoculum of 0.1% v / v and activated overnight at 37°C and 200 r / min to obtain seed culture. The seed culture was then transferred to LB medium containing streptomycin at an inoculum of 2% v / v and cultured at 37°C and 200 r / min until OD600 = 0.6–0.8. IPTG was then added to a final concentration of 0.2 mmol / L for induction, and fermentation was carried out at 30°C and 200 r / min for 12 h. The content of 5-aminolevulinic acid (5-ALA) was measured, and the results are shown in Table 2.

[0032] Table 2 5-ALA Measurement Results

[0033] The results showed that the 5-ALA content of strain WTΔA-pC / C398A after fermentation was 24.54 mg / L, which was lower than that of WTΔA-pC / A. R It increased by 42%.

[0034] Example 3: Expression of ferrous chelate gene hemH Ferrous chelate FeCH hemH The gene (the amino acid sequence of ferrous chelate, shown in SEQ ID NO: 6) was ligated between the Nde I and Xho I sites of plasmid pC / C398A (prepared in Example 2) to obtain plasmid pC / C398AH. Plasmid pC / C398AH was then transformed into strain WTΔA to obtain strain WTΔA-pC / C398AH. The technical route of this application is as follows: Figure 1 As shown.

[0035] Example 4: Determination of heme production by bacterial strain during fermentation strains WT, WTΔA-pC / A R WTΔA-pC / C398A and WTΔA-pC / C398AH were inoculated into LB medium containing streptomycin at an inoculum of 0.1% v / v and activated overnight at 37°C and 200 r / min to obtain seed culture. The seed culture was then transferred to LB medium containing streptomycin at an inoculum of 2% v / v and cultured at 37°C and 200 r / min until OD600 = 0.6~0.8. IPTG was then added to a final concentration of 0.2 mmol / L for induction, and fermentation was carried out at 30°C and 200 r / min for 12 h. The heme content was measured, and the results are shown in Table 3.

[0036] Table 3 Heme Measurement Results

[0037] After fermentation, the heme content of strain WTΔA-pC / C398AH reached 209.63 μmol / L, which was 47.20% higher than that of WTΔA-pC / C398A.

[0038] Example 5: Fermentation with exogenous L-Gly The strain WTΔA-pC / C398AH was subjected to shake-flask fermentation using the method described in Example 4, the difference being that 50, 100, 200, 300, and 400 mg / L of L-Gly were added exogenously during the fermentation process; the heme content was measured, and the results are shown in Table 4. The results showed that when 200 mg / L of L-Gly was added exogenously, the heme content of strain WTΔA-pC / C398AH increased to 370.18 μmol / L.

[0039] Table 4

[0040] The sequences involved in the embodiments are as follows: The amino acid sequence of aminoacyl-tRNA reductase (GluTR) is shown in SEQ ID NO: 1: MTLLALGINHKTAPVSLRERVSFSPDKLDQALDSLLAQPMVQGGVVLSTCNRTELYLSVEEQDNLQEALIRWLCDYHNLNEEDLRKSLYWHQDNDAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQKGHMKASELERMFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESLSTVTVLLVGAGETIELVARHLREHKVQKMIIANRTRERAQILADEVGAEVIALSEIDERLREADIIISSTASPLPIIGKGMVERALKSRRNQPMLLVDIAVPRDVEPEVGKLANAYLYSVDDLQSIISHNLAQRKAAAVEAETIVAQETSEFMAWLRAQSASETIREYRSQAEQVRDELTAKALAALEQGGDAQAIMQDLAWKLTNRLIHAPTKSLQQAARDGDNERLNILRDSLGLE Rs The amino acid sequence of ALAS is shown in SEQ ID NO: 2: MDYNLALDTALNRLHTEGRYRTFIDIERRKGAFPKAMWRKPDGSEKEITVWCGNDYLGMGQHPVVLGAMHEALDSTGAGSGGTRNISGTTLYHKRLEAELADLHGKEAALVFSSAYIANDATLSTLPQLIPGLVIVSDKLNHASMIEGIRRSGTEKHIFKHNDLDDLRRILTSIGKDRPILVAFESVYSMDGDFGRIEEICDIADEFGALKYIDEVHAVGMYGPRGGGVAERDGLMDRIDIINGTLGKAYGVFGGYIAASSKMCDAVRSYAPGFIFSTSLPPVVAAGAAASVRHLKGDVELREKHQTQARILKMRLKGLGLPIIDHGSHIVPVHVGDPVHCKMISDMLLEHFGIYVQPINFPTVPRGTERLRFTPSPVHDSGMIDHLVKAMDVLWQHCALNRAEVVA The amino acid sequence of mutant C264A is shown in SEQ ID NO: 3 MDYNLALDTALNRLHTEGRYRTFIDIERRKGAFPKAMWRKPDGSEKEITVWCGNDYLGMGQHPVVLGAMHEALDSTGAGSGGTRNISGTTLYHKRLEAELADLHGKEAALVFSSAYIANDATLSTLPQLIPGLVIVSDKLNHASMIEGIRRSGTEKHIFKHNDLDDLRRILTSIGKDRPILVAFESVYSMDGDFGRIEEICDIADEFGALKYIDEVHAVGMYGPRGGGVAERDGLMDRIDIINGTLGKAYGVFGGYIAASSKMADAVRSYAPGFIFSTSLPPVVAAGAAASVRHLKGDVELREKHQTQARILKMRLKGLGLPIIDHGSHIVPVHVGDPVHCKMISDMLLEHFGIYVQPINFPTVPRGTERLRFTPSPVHDSGMIDHLVKAMDVLWQHCALNRAEVVA The amino acid sequence of mutant C341A is shown in SEQ ID NO: 4 MDYNLALDTALNRLHTEGRYRTFIDIERRKGAFPKAMWRKPDGSEKEITVWCGNDYLGMGQHPVVLGAMHEALDSTGAGSGGTRNISGTTLYHKRLEAELADLHGKEAALVFSSAYIANDATLSTLPQLIPGLVIVSDKLNHASMIEGIRRSGTEKHIFKHNDLDDLRRILTSIGKDRPILVAFESVYSMDGDFGRIEEICDIADEFGALKYIDEVHAVGMYGPRGGGVAERDGLMDRIDIINGTLGKAYGVFGGYIAASSKMCDAVRSYAPGFIFSTSLPPVVAAGAAASVRHLKGDVELREKHQTQARILKMRLKGLGLPIIDHGSHIVPVHVGDPVHAKMISDMLLEHFGIYVQPINFPTVPRGTERLRFTPSPVHDSGMIDHLVKAMDVLWQHCALNRAEVVA The amino acid sequence of mutant C398A is shown in SEQ ID NO: 5 MDYNLALDTALNRLHTEGRYRTFIDIERRKGAFPKAMWRKPDGSEKEITVWCGNDYLGMGQHPVVLGAMHEALDSTGAGGSGGTRNISGTTLYHKRLEAELADLHGKEAALVFSSAYIANDATLSTLPQLIPGLVIVSDKLNHASMIEGIRRSGTEKHIFKHNDLDDLRRILTSIGKDRPILVAFESVYSMDGDFGRIEEICDI ADEFGALKYIDEVHAVGMYGPRGGGVAERDGLMDRIDIINGTLGKAYGVFGGYIAASSKMCDAVRSYAPGFIFSTSLPPVVAAGAAASVRHLKGDVELREKHQTQARILKMRLKGLGLPIIDHGSHIVPVHVGDPVHCKMISDMLLEHFGIYVQPINFPTVPRGTERLRFTPSPVHDSGMIDHLVKAMDVLWQHAALNRAEVVA The amino acid sequence of ferrochelase is shown in SEQ ID NO: 6. MRQTKTGILLANLGTPDAPTPEAVKRYLKQFLSDRRVVDTSRLLWWPLLRGVILPLRSPRVAKLYASVWMEGGSPLMVYSRQQQQALAQRLPETPVALGMSYGSPSLESAVDELLAEHVDHIVVLPLYPQYSCSTVGAVWDELARILARKRSIPGISFIR DYADNHDYINALANSVRASFAKHGEPDLLLLSYHGIPQRYADEGDDYPQRCRTTTRELASALGMAPEKVMMTFQSRFGREPWLMPYTDETLKMLGEKGVGHIQVMCPGFAADCLETLEEIAEQNREVFLGAGGKKYEYIPALNATPEHIEMMANLVAAYR Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant Escherichia coli that synthesizes heme, characterized in that, The *E. coli* strain had its gene encoding aminoacyl-tRNA reductase (GluTR) knocked out and overexpressed [the enzyme]. Rs ALAS or its mutants; the Rs The amino acid sequence of ALAS is shown in SEQ ID NO: 2; the mutant is the one with the amino acid sequence shown in SEQ ID NO:

2. Rs The ALAS was obtained by mutating cysteine ​​at position 398 to alanine.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The *E. coli* strain simultaneously overexpressed ferrous chelate; the amino acid sequence of the ferrous chelate is shown in SEQ ID NO:

6.

3. The recombinant Escherichia coli according to claim 1, characterized in that, The amino acid sequence of the aminoacyl-tRNA reductase (GluTR) is shown in SEQ ID NO:

1.

4. A 5-aminolevulinic acid synthase mutant, characterized in that, The mutant is one whose amino acid sequence is shown in SEQ ID NO:

2. Rs The ALAS was obtained by mutating cysteine ​​at position 398 to alanine.

5. A gene encoding the 5-aminolevulinic acid synthase mutant of claim 4 or a recombinant vector carrying the gene.

6. A method for increasing the yield of heme in Escherichia coli, characterized in that, The *E. coli* strain had its gene encoding aminoacyl-tRNA reductase (GluTR) knocked out and overexpressed ferrous chelase. Rs ALAS or its mutants; The amino acid sequence of the aminoacyl-tRNA reductase (GluTR) is shown in SEQ ID NO: 1; Rs The amino acid sequence of ALAS is shown in SEQ ID NO: 2; the mutant is the one with the amino acid sequence shown in SEQ ID NO:

2. Rs The ALAS was obtained by mutating cysteine ​​at position 398 to alanine; the amino acid sequence of the ferrous chelate is shown in SEQ ID NO:

6.

7. A method for producing heme, characterized in that, The method includes fermentation production using recombinant Escherichia coli as described in any one of claims 1 to 3.

8. The method according to claim 7, characterized in that, The fermentation is inoculating the recombinant E. coli into the fermentation medium with the inoculation amount of 1-5% v / v, culturing at 30-37°C until OD 600 When OD is 0.6-0.8, IPTG with the final concentration of 0.1-0.5 mmol / L is added, and the induction is carried out at 16-37°C for 10-24 h.

9. The method according to claim 8, characterized in that, The fermentation medium may also contain L-Gly at a concentration of 50-400 mg / L.

10. The use of the recombinant Escherichia coli according to any one of claims 1 to 3 or the method according to claim 6 in the synthesis of heme or in the preparation of products containing heme.

Images