A method for constructing recombinant escherichia coli for efficient synthesis of hematin

Abstract

The application discloses a method for constructing recombinant escherichia coli for efficiently synthesizing hematin, and belongs to the technical field of biology. The hemL gene of the C5 pathway of the escherichia coli is knocked out, the 5-aminolevulinic acid (5-ALA) synthase gene of the C4 pathway is introduced, and the expression of hemB or hemH of the escherichia coli is strengthened, so that the hematin synthesis capacity is improved. The genetically engineered bacteria constructed in the application have a greatly improved hematin content compared with the starting strain, and the hematin concentration of the best one reaches 59.46 mu mol L ‑1 .

Description

Technical Field

[0001] This invention relates to a method for constructing recombinant Escherichia coli to efficiently synthesize heme, belonging to the field of biotechnology. Background Technology

[0002] Heme is a metalloporphyrin containing ferrous ions and is a high-quality iron source, which can be used as an iron supplement in food processing and manufacturing. Currently, the chemical synthesis and the production of heme using organic extraction or enzymatic hydrolysis of biomaterials are complex, low-yield, time-consuming, and environmentally unfriendly processes. Biosynthesis of heme using recombinant bacteria is the most promising method for large-scale heme production; however, current yields are low and unsuitable for industrial production. Therefore, it is essential to enhance the heme synthesis capacity of recombinant bacteria through metabolic engineering.

[0003] Heme synthesis in organisms can be divided into two pathways: C4 and C5, differing in the synthesis of the precursor 5-aminolevulinic acid (5-ALA). In the C4 pathway, ALA synthase (ALAS, hemA) catalyzes the condensation of L-glycine (L-Gly) and succinyl-CoA to form 5-ALA, requiring only one step. In the C5 pathway, glutamate-tRNA synthase (GluTS, gltX) catalyzes the formation of glutamyl-tRNA from L-glutamate (Glu), followed by glutamyl-tRNA reductase (GluTR, hemA) catalyzing the formation of glutamate-1-semialdehyde. The glutamate-1-semialdehyde-2,1-mutase (GSAM, hemL) then transaminates to 5-ALA, requiring three enzymatic steps. Generally, only one of the C4 or C5 pathways exists in an organism; for example, *Escherichia coli* synthesizes heme via the C5 pathway. The conversion of 5-ALA to heme requires seven steps, including the asymmetric condensation of two molecules of 5-ALA by pyrrole derivative pyrrobilinogen (PBG) encoded by hemeB, and the ferrous chelate enzyme (hemH) that chelates Fe... 2+ The key step is the insertion of protoporphyrin IX (PPIX) to form heme.

[0004] Currently, improving the heme synthesis capacity of E. coli through metabolic engineering is mainly based on the heme synthesis pathway, specifically including: 1) increasing the supply of precursors, such as overexpression of GluTS to increase the level of 5-ALA, but GluTS is subject to feedback control by the final product heme, and the overexpression of a single gene can affect the expression level of other genes in the pathway; 2) modularizing the pathway and adjusting the gene expression intensity between modules, involving multiple genes and multiple vectors, which increases the metabolic burden on the host; 3) introducing the C4 pathway, but since the C5 and C4 pathways coexist, the two pathways will compete with each other, affecting the synthesis of the final product heme.

[0005] Therefore, it is necessary to develop more different types of recombinant Escherichia coli to further improve heme synthesis. Summary of the Invention

[0006] To address at least one of the aforementioned problems, this invention modifies the inherent C5 pathway of *E. coli* to a C4 pathway by blocking the C5 pathway and introducing the ALAS gene from *Rhodotorula spheroides*, instead of a coexistence of C4 and C5. Simultaneously, it enhances the expression of key genes *hemB* or *hemH*, thereby increasing heme production. This invention knocks out the *hemL* gene in the *E. coli* C5 pathway and introduces the *hemA* gene from *Rhodotorula spheroides*' ALAS pathway. R Furthermore, by overexpressing the genes hemeB or hemeH, which are involved in the synthesis of heme from 5-ALA, a recombinant Escherichia coli that synthesizes heme efficiently was constructed.

[0007] The first objective of this invention is to provide a recombinant *E. coli* strain that efficiently synthesizes heme, wherein the recombinant *E. coli* strain has the *hemL* gene encoding glutamate-1-semialdehyde-2,1-aminotransmutase knocked out or silenced, and overexpresses the *hemA* gene of *Rhodopseudomonas spheroidae*. R .

[0008] In one embodiment, the recombinant Escherichia coli also overexpresses the hemH gene encoding Escherichia coli ferrous chelate.

[0009] In one embodiment, the recombinant Escherichia coli also overexpresses the hemB gene encoding Escherichia coli bile pigment synthase.

[0010] In one embodiment, the amino acid sequence encoded by the hemL gene is the sequence CAQ30669.1 in GenBank on NCBI; hemA R The amino acid sequences encoded by the hemH gene are those in GenBank AAA72325.1, the amino acid sequences encoded by the hemH gene are those in GenBank CAQ30948.1, and the amino acid sequences encoded by the hemB gene are those in GenBank CAQ30840.1.

[0011] In one implementation, the hemL gene is knocked out using Red homologous recombination.

[0012] In one implementation, hemA R The hemH or hemB gene is expressed in the host via plasmids.

[0013] In one implementation, the host is Escherichia coli BL21(DE3).

[0014] In one embodiment, the plasmid vector is pCDFDuet1.

[0015] A second objective of this invention is a method for increasing heme production in *Escherichia coli*, the method comprising knocking out or silencing the *hemL* gene encoding glutamate-1-semialdehyde-2,1-aminotransmutase, and overexpressing the *hemA* gene of *Rhodopseudomonas spheroidae*. R .

[0016] In one embodiment, the method overexpresses the hemH gene encoding ferrous chelate synthase or the hemB gene encoding bile pigmentogen synthase.

[0017] A third objective of this invention is to provide a method for producing heme, the method comprising fermentation production using the recombinant Escherichia coli of this invention.

[0018] In one embodiment, the fermentation involves inoculating the recombinant bacteria into the fermentation medium at an inoculum rate of 1–5%, culturing at 30–37°C until the OD600 reaches 0.6–0.8, and then adding a final concentration of 0.1–0.5 mmol·L⁻¹. -1 IPTG induction culture for 8–16 h.

[0019] A fourth objective of this invention is to provide the application of the recombinant Escherichia coli or the above-described method for increasing the heme production of Escherichia coli in the synthesis of heme.

[0020] Beneficial effects:

[0021] This invention constructs a C4 pathway by knocking out the hemL gene in the C5 pathway of *E. coli* and simultaneously introducing 5-aminolevulinic acid (5-ALA) synthase from *Rhodotorula spheroides*, thereby enhancing the expression of *hemB* or *hemH* in *E. coli* to increase heme production. Compared to the starting strain, the genetically engineered bacteria constructed in this invention exhibit a significantly increased heme content, with the optimal heme concentration reaching 59.46 μmol·L⁻¹. -1 . Attached Figure Description

[0022] Figure 1 This is a schematic diagram of heme metabolism modification. Detailed Implementation

[0023] 1. LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0.

[0024] 2. Heme concentration determination:

[0025] Heme concentration was detected using a fluorescence method, specifically by taking an appropriate amount of cultured bacterial cells and adjusting the OD value. 600× bacterial volume (mL) = 8 (for example: if the OD of the bacterial cells 600 If the value is 0.4, take 20 mL of bacterial culture and centrifuge at 12000 r / min for 5 min at 4℃ to obtain bacterial precipitate. After washing with water, 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 overnight at 4℃ in the dark 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 it at room temperature as an experimental control group. Take the other half of the sample (for porphyrin and total heme concentration detection) and heat it at 95℃ for 30 min. After natural cooling, centrifuge at 12000 r / min for 5 min. Take 200 μL to a black 96-well plate and perform fluorescence value detection (excitation wavelength 400 nm, emission wavelength 620 nm). The difference between the two groups is the heme concentration to be tested.

[0026] 3. Preparation of competent E. coli cells: Refer to the instructions for the Takara Competent Cell Preparation Kit.

[0027] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0028] Example 1: Knockout of the hemL gene and expression of hemA R Recombinant E. coli WTΔL / pCA gene R

[0029] Based on the E. coli BL21(DE3) genome published on NCBI (https: / / www.ncbi.nlm.nih.gov / nuccore / AM946981.2), upstream and downstream primers for knocking out the hemL gene homologous sequences were designed, along with identification primers. The hemL gene (encoding the amino acid sequence CAQ30669.1 in GenBank on NCBI) in *E. coli* BL21(DE3) (named WT) was knocked out using 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 for details). Sequencing confirmed the presence of the knocked-out hemL gene in the genetically engineered bacterium, which was named WTΔL.

[0030] The ALAS synthase gene hemA from the C4 pathway R (The encoded amino acid sequence is the sequence AAA72325.1 in GenBank on NCBI.) It is ligated to the NdeI and EcoRV restriction sites of the vector pCDFDuet1 to form the plasmid pCA. R The strain was introduced into WTΔL competent cells and named WTΔL / pCA. R .

[0031] Starting strain WT and WTΔL / pCA R The culture medium was inoculated into shake flasks containing 50 mL of LB liquid medium and activated overnight at 37 °C and 200 rpm. Then, 2% of the inoculum was added to 150 mL of LB liquid medium and cultured for 12 h. For the plasmid-containing strain, kanamycin was added, and after 2 h of culture, IPTG at a final concentration of 0.2 mmol / L was added for induction for 8 h. WTΔL / pCA R The strain's heme content reached 43.51 μmol / L, which is 4.32 times that of WT.

[0032] Example 2: Overexpression of hemA R Recombinant Escherichia coli WTΔL / pCHA with hemH R

[0033] The *E. coli* ferrochelate synthase gene *hemH* (encoding the amino acid sequence CAQ30948.1 from GenBank, NCBI) was ligated into plasmid pCA. R The plasmid pCHA is formed. R And introduced into WTΔL competent cells to obtain co-overexpressing hemA R The recombinant strain WTΔL / pCHA with hemH R .

[0034] Same as in Example 1, shake flask culture was performed, and the heme concentration reached 59.46 μmol·L⁻¹. -1 .

[0035] Example 3: Overexpression of hemA R Recombinant E. coli WTΔL / pCBA with hemB gene R

[0036] The bile chromogen synthase gene hemB (the amino acid sequence encoded by the hemB gene is CAQ30840.1 in GenBank, NCBI) was ligated into the plasmid pCA. R plasmid pCBA was obtained. R And introduced into WTΔL competent cells to obtain co-overexpressing hemA RThe recombinant strain WTΔL / pCBA with hemB R .

[0037] Same as in Example 1, shake flask culture was performed, and the heme concentration reached 46.84 μmol·L⁻¹. -1 .

[0038] 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 recombinant E. coli knocked out or silenced the enzyme encoding glutamate-1-semialdehyde-2,1-aminomutase. hemL Genes, and overexpression of the ALAS gene of Rhodops spheroidae. hemA R The recombinant Escherichia coli also overexpressed an enzyme encoding Escherichia coli ferrous chelase. hemH Genes, or those encoding Escherichia coli bile pigment synthase. hemB Genes; the stated hemL The amino acid sequence encoded by the gene is the sequence CAQ30669.1 in GenBank on NCBI; hemA R The amino acid sequence encoded by the gene is the sequence AAA72325.1 in GenBank on NCBI. hemH The amino acid sequence encoded by the gene is the sequence CAQ30948.1 in GenBank on NCBI; hemB The amino acid sequence encoded by the gene is the sequence CAQ30840.1 in GenBank on NCBI.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The hemL The gene was knocked out using the Red homologous recombination method.

3. The recombinant Escherichia coli according to claim 1, characterized in that, The recombinant Escherichia coli was constructed based on Escherichia coli BL21(DE3).

4. A method for increasing the heme production of Escherichia coli, characterized in that, The method involves knocking out or silencing the enzyme encoding glutamate-1-semialdehyde-2,1-aminomutase in the *E. coli* genome. hemL Genes, and overexpression of the ALAS gene in Rhodops spheroidae. hemA R The method also overexpresses the enzyme encoding ferrous chelase. hemH Genes or those encoding bile pigment synthase hemB Genes, the ones mentioned hemL The amino acid sequence encoded by the gene is the sequence CAQ30669.1 in GenBank on NCBI; hemA R The amino acid sequence encoded by the gene is the sequence AAA72325.1 in GenBank on NCBI. hemH The amino acid sequence encoded by the gene is the sequence CAQ30948.1 in GenBank on NCBI; hemB The amino acid sequence encoded by the gene is the sequence CAQ30840.1 in GenBank on NCBI.

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

6. The method according to claim 5, characterized in that, The fermentation process involves inoculating the recombinant bacteria into the fermentation medium at an inoculum rate of 1-5%, culturing at 30-37ºC until the OD600 reaches 0.6-0.8, and then adding 0.1-0.5 mmol·L⁻¹ to a final concentration. -1 IPTG induction culture for 8-16 h.

7. The application of the recombinant Escherichia coli according to any one of claims 1-3, the method for increasing the heme production of Escherichia coli according to claim 4, or the method for producing heme according to claim 5 or 6 in the synthesis of heme.

Images