Recombinant escherichia coli for producing heme and derivative compounds thereof and preparation method of recombinant escherichia coli

Abstract

The invention discloses recombinant escherichia coli for producing heme and derivative compounds thereof and a preparation method of the recombinant escherichia coli. According to the invention, on the basis of escherichia coli, ptsG, galR, lpxM, pta, poxB, arcA, ldhA, sdhA, iclR, metA and dapD are knocked out; the method comprises the following steps: introducing glk, Zglf, acs, ppc, BsmetA, BsyjcI, BsdapH, BspatA and BscdapL, and enhancing the expression of araE, stA, coA, serA and gltA; and the expression of icd is weakened. And the expression of eamA and rhtA is enhanced. The GYDH and the hemA are introduced; according to the recombinant strain, D-glucose is used as a raw material to synthesize 5-ALA, and on the basis of the 5-ALA, coding genes of the heme and the various derivative compounds thereof are introduced, so that recombinant bacteria for producing the heme and the various derivative compounds thereof can be obtained.

Description

Technical Field

[0001] The present invention belongs to the fields of synthetic biology, microbial genome modification and metabolic regulation, and food and pharmaceutical raw material chemical industry, and specifically relates to recombinant Escherichia coli for producing heme and its derivative compounds and a preparation method thereof. Background Art

[0002] The artificial meat market has developed rapidly in recent years and has become a hot topic in the field of food technology. Artificial meat is based on plant protein or cell culture technology and aims to provide a more sustainable, healthy and ethical meat substitute. Heme, as one of the key ingredients of artificial meat, plays an important role in improving taste, flavor and texture. By binding to the protein components of artificial meat, heme can give artificial meat a "meaty aroma" and rosy appearance similar to traditional meat, making the consumer experience more realistic. As consumers pay more attention to environmental protection, healthy eating and animal welfare, the artificial meat market is expected to continue to expand. However, the cost control, consumer acceptance and regulatory approval of heme are still challenges facing the industry. In the future, with technological advancements and the realization of large-scale production, artificial meat and its core ingredient heme are expected to further promote the transformation and upgrading of the food industry and provide the world with more sustainable dietary options. Heme is a Fe-containing 2+ Porphyrin compounds play an important role in regulating iron balance, transporting oxygen, transferring electrons, and providing energy, and are widely used in the food and healthcare industries. Heme derivatives also have a wide range of applications in medicine, healthcare, food, and other fields.

[0003] Heme production primarily relies on chemical synthesis or extraction from plant tissues and animal blood. These methods yield products of low purity, produce numerous byproducts, and pose significant environmental risks. Several high-value-added derivatives of heme occur naturally, but their yields are extremely low, resulting in high extraction costs that far exceed market demand. In recent years, biosynthesis has seen rapid development in the production of various high-value-added chemicals. Biosynthesis utilizes abundant natural raw materials and enzymes as catalysts to produce a variety of products. This not only reduces industrial production costs but also adheres to current green and environmentally friendly production principles. Glucose is an inexpensive, readily available, green organic raw material that provides additional reducing power for biosynthesis, thereby increasing conversion rates in biomanufacturing, making it an ideal raw material for biomanufacturing. Therefore, constructing cell factories that initially grow on glucose and then synthesize various chemicals using glucose as a feedstock has significant economic and social value.

[0004] The metabolic pathway for de novo heme synthesis using glucose as a raw material in microbial cell factories is divided into upstream and downstream pathways with 5-aminolevulinic acid (5-ALA) as the dividing point. The upstream pathway mainly refers to the conversion of glucose to various intermediate metabolites through the tricarboxylic acid cycle, and then to 5-aminolevulinic acid (5-ALA) through the C4 pathway with glycine as a precursor or the C5 pathway with glutamate as a precursor. The downstream pathway refers to the synthesis of heme using 5-ALA as the sole precursor, through three pathways in different organisms: coproporphyrin-dependent, protoporphyrin-dependent, and siroheme-dependent. Heme can be secreted outside the cell through transport proteins to become the final product; it can also be used as a substrate inside the cell and continue to generate a series of high-value-added heme-derived compounds such as biliverdin, phycocyanin, and bilirubin through different metabolic pathways.

[0005] 5-ALA is the common precursor of all natural tetrapyrrole compounds and the only source of carbon and nitrogen atoms necessary for the production of heme and its derivatives. Therefore, high-yield 5-ALA is a prerequisite for the large-scale production of heme and its derivatives using microorganisms. Domestic and foreign researchers have conducted a series of studies on how to efficiently synthesize 5-ALA. According to published research data, there are several major bottlenecks in the efficient synthesis and accumulation of 5-ALA using biological methods: (1) Whether it is the C4 pathway or the C5 pathway, the important precursors for the synthesis of 5-ALA come from the tricarboxylic acid cycle. The metabolic pathway is relatively complex, with many branches and a long pathway, resulting in extremely low yields, which limits the production of 5-ALA downstream. (2) 5-ALA is easily degraded in the intracellular environment. (3) Excessive accumulation of 5-ALA in cells can damage cells, affect their normal growth and metabolism, and produce feedback inhibition on the synthesis pathway.

[0006] The downstream synthesis pathway from 5-ALA to heme also faces numerous limitations, including: 1) low conversion efficiency due to low activity of certain enzymes in the pathway; 2) carbon flux loss due to excessive bypass; and 3) cellular toxicity and feedback inhibition of the metabolic pathway by heme. Furthermore, the synthesis of related derivatives using heme as a substrate is primarily limited by the heterologous expression of key enzymes and their impact on normal cellular metabolism.

[0007] There has been a lot of work on metabolic engineering of microbial heme and its derivatives. However, due to the lack of effective regulation and optimization strategies, it is currently impossible to synthesize heme and its derivatives at high levels in microbial strains. The yield of heme and its derivatives is low (heme yield does not exceed 300 mg / L (Nature Catalysis V1:720-728; CN106434509A); ​​the heme derivative bilirubin does not exceed 20 mg / L (CN116555148A). In actual experiments, expensive intermediates such as glutamate, glycine, and succinate must be added, making these technologies difficult to implement. A key factor in synthesizing heme and its derivatives using glucose as the sole raw material is that during the coordinated expression of different genes, the metabolic pathway is blocked in the intermediate catalytic process due to flux imbalance. For example, when synthesizing 5-ALA precursors via the C4 pathway, the imbalance between the succinyl-CoA and glycine synthesis pathways can reduce synthesis efficiency. During the synthesis of heme and heme derivatives, the supply and release of iron ions significantly affect the related catalytic reactions. To address these issues, it is necessary to utilize protein expression regulation to simultaneously control the expression levels of multiple genes to achieve a balanced intensity of key reactions, thereby improving synthesis flux.

[0008] While these static regulation methods can balance metabolic flux and improve synthesis efficiency to a certain extent, their imprecision still results in significant energy loss and metabolic imbalance, limiting the production of heme and its derivatives. Excessive use of plasmids for overexpression can also affect normal cell growth and production stability. These factors limit the commercialization value of biosynthetic production of heme and its derivatives. Summary of the Invention

[0009] The technical problem to be solved by the present invention is: how to achieve a dynamic balance of metabolic flow in Escherichia coli cells so that they can efficiently synthesize 5-ALA using glucose as a raw material, and use it as an important precursor to synthesize hemoglobin and its various derivative compounds.

[0010] In order to solve the above technical problems, in a first aspect, the present invention provides a recombinant Escherichia coli, which is a recombinant bacterium obtained by carrying out the following transformations 1)-23) and 26) in Escherichia coli;

[0011] 1) Knockout of the ptsG gene encoding the phosphotransferase G subunit;

[0012] 2) Introducing the glucokinase encoding gene glk and its expression regulatory factors;

[0013] 3) Knockout of the galactose repressor gene galR;

[0014] 4) Introducing the gene encoding the glucose transport cofactor Zglf and its expression regulator;

[0015] 5) Knockout of the gene lpxM encoding myristoyltransferase involved in lipid A biosynthesis;

[0016] 6) Enhance the expression of araE, the gene encoding the arabinose transporter;

[0017] 7) Enhancing the expression of sthA, the gene encoding NAD(P) transhydrogenase;

[0018] 8) Knockout the gene encoding phosphate acetyltransferase, pta;

[0019] 9) Introducing the acetyl-CoA synthetase encoding gene acs and its expression regulatory factors;

[0020] 10) Knockout of the gene encoding pyruvate oxidase, poxB;

[0021] 11) Knockout of the gene encoding the transcriptional regulator arcA;

[0022] 12) Knockout of the lactate dehydrogenase encoding gene ldhA;

[0023] 13) Enhance the expression of the gene encoding pantothenate kinase, coaA;

[0024] 14) Enhancing the expression of serA, the gene encoding phosphoglycerate dehydrogenase;

[0025] 15) Knockout of the sdhA gene encoding the A subunit of succinate dehydrogenase;

[0026] 16) Enhancing the expression of the gene gltA encoding citrate synthase;

[0027] 17) Weakened expression of the gene encoding isocitrate dehydrogenase, icd;

[0028] 18) Knockout of the gene encoding isocitrate lyase inhibitor iclR;

[0029] 19) Introducing the ppc gene encoding phosphoenolpyruvate carboxylase and its expression regulatory factor;

[0030] 20) Knockout of metA, the gene encoding homoserine succinyltransferase;

[0031] 21) introducing the homoserine acetyltransferase encoding gene BsmetA and the acetylhomoserine lyase encoding gene BsyjcI and their expression regulatory factors;

[0032] 22) Knockout of the dapD gene encoding tetrahydrodipyridine N-succinate transferase;

[0033] 23) introducing the genes encoding tetrahydrodipyridine N-acetyltransferase BsdapH, N-acetyldiaminopimelate aminotransferase BspatA, and N-acetyldiaminopimelate deacetylase BscdapL, as well as their expression regulatory factors;

[0034] 26) The gene encoding glycine dehydrogenase, GYDH, and the gene encoding 5-aminolevulinic acid synthase, hemA, were introduced.

[0035] In the above-mentioned recombinant bacteria, the modification further comprises 24) enhancing the expression of the gene eamA encoding the cysteine / O-acetylserine efflux protein;

[0036] 25) Enhance the expression of rhtA, a gene encoding a threonine / homoserine efflux protein.

[0037] The above modifications 1) and 2) can be specifically: replacing the gene ptsG encoding the phosphotransferase subunit G with the constitutive promoter P 119 and the gene glk encoding glucokinase (this modification corresponds to HA101 in the example).

[0038] The above modifications 3) and 4) can be specifically: replacing the gene galR encoding the galactose repressor with the constitutive promoter P CPA1 and encoding glucose transport cofactors (galactose: + The symporter gene Zglf was modified (this modification corresponds to HA102 in the example).

[0039] The above modification 6) can be specifically: replacing the promoter of the arabinose transporter gene araE with the Escherichia coli constitutive promoter P CPA1 (This modification corresponds to HA104 of the embodiment).

[0040] The above modification 7) can be specifically: replacing the promoter of the NAD(P) transhydrogenase gene sthA with the Escherichia coli constitutive promoter P 119 (This modification corresponds to HA105 of the embodiment).

[0041] The above modifications 8) and 9) can be specifically: replacing the gene pta encoding phosphate acetyltransferase with the constitutive promoter P CPA1 and the gene acs encoding acetyl-CoA synthetase (this modification corresponds to HA106 in the example).

[0042] The above modification 13) can be specifically: replacing the pantothenate kinase (coaA) gene promoter with the Escherichia coli constitutive promoter P CPA1 (This modification corresponds to HA110 of the embodiment in turn).

[0043] The above modification 14) can be specifically: replacing the phosphoglycerate dehydrogenase (serA) gene promoter with the Escherichia coli constitutive promoter P CPA1 (This modification corresponds to HA111 of the embodiment in turn).

[0044] The above modification 16) can be specifically: replacing the promoter of the citrate synthase gltA gene with the Escherichia coli constitutive promoter P 119 (This modification corresponds to HA113 of the embodiment).

[0045] The above modification 17) can be specifically: replacing the isocitrate dehydrogenase icd gene promoter with the Escherichia coli constitutive promoter P 113 (This modification corresponds to HA114 of the embodiment).

[0046] The above modifications 18) and 19) can specifically be to replace the isocitrate lyase repressor gene iclR with the constitutive promoter P 119 and the gene ppc encoding phosphoenolpyruvate carboxylase (this modification corresponds to HA115 in the example).

[0047] The above modifications 20) and 21) can be specifically: replacing the gene metA encoding homoserine succinyltransferase with the constitutive promoter P CPA1 and the gene encoding homoserine acetyltransferase BsmetA and the gene encoding acetylhomoserine lyase BsyjcI (this modification corresponds to HA116 in the example).

[0048] The above modifications 22) and 23) can be specifically: replacing the gene dapD encoding tetrahydrodipyridine N-succinate transferase with the constitutive promoter P CPA1 , a gene BsdapH encoding tetrahydrodipyridine N-acetyltransferase, a gene BspatA encoding N-acetyldiaminopimelate aminotransferase, and a gene BscdapL encoding N-acetyldiaminopimelate deacetylase (this modification corresponds to HA117 in the example).

[0049] Above transformation:

[0050] 24) Enhance the expression of the cysteine / O-acetylserine efflux protein gene eamA;

[0051] 25) Enhancing the expression of the threonine / homoserine efflux protein gene rhtA;

[0052] The above modification 24) can be specifically: replacing the promoter of the cysteine / O-acetylserine efflux protein eamA gene with the Escherichia coli constitutive promoter P CPA1 (This modification corresponds to HA118 of the embodiment in turn).

[0053] The above modification 25) can be specifically: replacing the promoter of the threonine / homoserine efflux protein rhtA gene with the Escherichia coli constitutive promoter P CPA1 (This modification corresponds to HA119 of the embodiment in turn).

[0054] The above modification 26) described in which the gene encoding glycine dehydrogenase GYDH and the gene encoding 5-aminolevulinic acid synthase hemA are introduced via the fusion fragment GYDH-RBS-hemA;

[0055] The fusion fragment GYDH-RBS-hemA includes a gene GYDH encoding glycine dehydrogenase, a gene hemA encoding 5-aminolevulinic acid synthase and an RBS connecting the two through the fusion fragment.

[0056] Specifically, glycine dehydrogenase GYDH and 5-aminolevulinic acid synthase hemA can be introduced in the form of plasmid pSB1s-GYDH-hemA.

[0057] The recombinant bacteria obtained by transformation in 1)-26) above correspond to HA121 in the example.

[0058] The recombinant bacteria obtained by transformation in 1)-23) and 26) above correspond to HA120B in the example.

[0059] In a second aspect, the present invention provides a recombinant Escherichia coli, comprising the modifications 1)-23), 26) and the following modifications in Escherichia coli:

[0060] 27) Knockout of the gene eamA encoding cysteine / O-acetylserine efflux protein;

[0061] 28) Knockout of the gene rhtA encoding threonine / homoserine efflux protein;

[0062] 29) Knockout of the porphyrinogen peroxidase encoding gene yfeX;

[0063] 30) Enhance the expression of hemB, the gene encoding porphobilinogen synthase;

[0064] 31) Enhancing the expression of the gene hemC encoding hydroxymethylcholine synthase;

[0065] 32) Enhancing the expression of hemE, the gene encoding uroporphyrinogen decarboxylase;

[0066] 33) Enhancing the expression of the gene hemF encoding coproporphyrinogen oxidase;

[0067] 34) Enhancing the expression of hemG, the gene encoding protoporphyrinogen oxidase;

[0068] 35) Introduce the ferrochelatase encoding gene hemH and the heme efflux protein gene cluster CcmABC.

[0069] In the above-mentioned recombinant Escherichia coli, the deposit number of the recombinant Escherichia coli is CGMCC No.32252.

[0070] In a third aspect, the present invention provides a method of performing the transformations 1) to 23), 26) and the following transformations in Escherichia coli:

[0071] 27) Knockout of the gene eamA encoding cysteine / O-acetylserine efflux protein;

[0072] 28) Knockout of the gene rhtA encoding threonine / homoserine efflux protein;

[0073] 29) Knockout of the porphyrinogen peroxidase encoding gene yfeX;

[0074] 30) Enhance the expression of hemB, the gene encoding porphobilinogen synthase;

[0075] 31) Enhancing the expression of the gene hemC encoding hydroxymethylcholine synthase;

[0076] 32) Enhancing the expression of hemE, the gene encoding uroporphyrinogen decarboxylase;

[0077] 33) Enhancing the expression of the gene hemF encoding coproporphyrinogen oxidase;

[0078] 34) Enhancing the expression of hemG, the gene encoding protoporphyrinogen oxidase;

[0079] 36) The gene encoding ferrochelatase, hemH, the gene encoding heme oxygenase, ho1, and the gene encoding glucose dehydrogenase, gdhA, were introduced.

[0080] In the above, the gene hemH encoding ferrochelatase, the gene ho1 encoding heme oxygenase, and the gene gdhA encoding glucose dehydrogenase described in 36) are introduced via a vector introducing a fusion fragment;

[0081] The fusion fragment comprises the gene hemH encoding ferrochelatase, the gene ho1 encoding heme oxygenase and a connecting peptide coding region connecting the two, and RBS and the gene gdhA encoding glucose dehydrogenase sequence.

[0082] In a fourth aspect, the present invention provides a recombinant Escherichia coli for carrying out the transformations 1)-23), 26) and the following transformations in the first aspect in Escherichia coli:

[0083] 27) Knockout of the gene eamA encoding cysteine / O-acetylserine efflux protein;

[0084] 28) Knockout of the gene rhtA encoding threonine / homoserine efflux protein;

[0085] 29) Knockout of the porphyrinogen peroxidase encoding gene yfeX;

[0086] 30) Enhance the expression of hemB, the gene encoding porphobilinogen synthase;

[0087] 31) Enhancing the expression of the gene hemC encoding hydroxymethylcholine synthase;

[0088] 32) Enhancing the expression of hemE, the gene encoding uroporphyrinogen decarboxylase;

[0089] 33) Enhancing the expression of the gene hemF encoding coproporphyrinogen oxidase;

[0090] 34) Enhancing the expression of hemG, the gene encoding protoporphyrinogen oxidase;

[0091] 36) The gene encoding ferrochelatase, hemH, and the gene encoding heme oxygenase, ho1, were introduced.

[0092] 37) Introduce the gene encoding biliverdin reductase bvr.

[0093] In the above-mentioned recombinant Escherichia coli, the deposit number of the recombinant Escherichia coli is CGMCC No.32251.

[0094] In a fifth aspect, the present invention provides recombinant Escherichia coli, which is used to carry out the transformations 1)-23), 26) and the following transformations in the first aspect in Escherichia coli:

[0095] 27) Knockout of the gene eamA encoding cysteine / O-acetylserine efflux protein;

[0096] 28) Knockout of the gene rhtA encoding threonine / homoserine efflux protein;

[0097] 29) Knockout of the porphyrinogen peroxidase encoding gene yfeX;

[0098] 30) Enhance the expression of hemB, the gene encoding porphobilinogen synthase;

[0099] 31) Enhancing the expression of the gene hemC encoding hydroxymethylcholine synthase;

[0100] 32) Enhancing the expression of hemE, the gene encoding uroporphyrinogen decarboxylase;

[0101] 33) Enhancing the expression of the gene hemF encoding coproporphyrinogen oxidase;

[0102] 34) Enhancing the expression of hemG, the gene encoding protoporphyrinogen oxidase;

[0103] 36) The gene encoding ferrochelatase, hemH, and the gene encoding heme oxygenase, ho1, were introduced.

[0104] 38) The pcyA gene encoding phycocyanin ferredoxin oxidoreductase was introduced.

[0105] The recombinant Escherichia coli mentioned above may be the recombinant bacterium with a preservation number of CGMCC No.32253.

[0106] The above modifications 27)-29) may specifically correspond to HA122, HA123 and HA124 of the embodiment in sequence.

[0107] The above transformations 30)-34) can be specifically to replace the promoters of each gene with the Escherichia coli constitutive promoter P in sequence. 119 , corresponding to HA125, HA126, HA127, HA128 and HA129 of the embodiment respectively.

[0108] The above modification 35) can be introduced in the form of pYB1k-hemH-CcmABC plasmid.

[0109] The recombinant bacteria obtained by transformation in 1)-23), 26), 27)-35) above correspond to HA130 in the examples.

[0110] The ferrochelatase encoding gene hemH and the heme oxygenase encoding gene ho1 described in modification 36) above can be introduced via the fusion fragment hemH-linker-ho1-RBS-gdhA;

[0111] The fusion fragment hemH-linker-ho1-RBS-gdhA includes the ferrochelatase encoding genes hemH and linker, the heme oxygenase encoding genes ho1 and RBS, and the glucose dehydrogenase encoding gene gdhA, and can be introduced in the form of the pYB1k2-hemH-ho1-gdhA plasmid.

[0112] The recombinant bacteria obtained by transformation in 1)-23), 26), 27)-34) and 36) above correspond to HA132 in the examples.

[0113] The above modifications 36) and 37) can be introduced in the form of pYB1k-hemH-ho1-bvr-gdhA plasmid.

[0114] The recombinant bacteria obtained by transformation of 1)-23), 26), 27)-34), 36), 37) above correspond to HA133 in the examples.

[0115] The above modifications 36) and 38) can be introduced in the form of pYB1k-HemH-ho1-pcyA-gdhA plasmid.

[0116] The recombinant bacteria obtained by transformation of 1)-23), 26), 27)-34), 36), and 38) above correspond to HA134 in the examples.

[0117] In the above text,

[0118] The expression regulating factors are factors that upregulate, promote or increase expression; specifically, they can be promoters.

[0119] Alternatively, the factor that upregulates, promotes or enhances expression is specifically a strong expression promoter.

[0120] Alternatively, in the modification, enhancing the expression of each gene is to replace the promoter of each gene with the strong expression promoter.

[0121] Alternatively, the weakening of gene expression is to replace the gene promoter with a weak expression promoter.

[0122] Or, the strong expression promoter is a constitutive promoter P CPA1 or constitutive promoter P 119。

[0123] Or, the weak expression promoter is a constitutive promoter P 113 .

[0124] Furthermore, the P CPA1 The nucleotide sequence is SEQ ID No.1.

[0125] Furthermore, the P 119 The nucleotide sequence is SEQ ID No.2.

[0126] Furthermore, the glucokinase (glk) may be derived from Escherichia coli.

[0127] Furthermore, the glucose transport cofactor (Zglf) may be derived from Zymomonas mobilis.

[0128] Furthermore, the acetyl-CoA synthetase (acs) may be derived from Escherichia coli.

[0129] Furthermore, the phosphoenolpyruvate carboxylase (ppc) may be derived from Escherichia coli.

[0130] Furthermore, the glycine dehydrogenase (GYDH) may be derived from Mycobacterium tuberculosis.

[0131] Furthermore, the ferrochelatase encoding gene hemH may be derived from Pantoea ananatis.

[0132] Furthermore, the 5-aminolevulinic acid synthase (hemA) may be derived from Rhodobacter capsulatus.

[0133] Furthermore, the 5-aminolevulinic acid synthase (hemA) may be derived from Rhodobacter capsulatus.

[0134] Furthermore, the heme oxygenase (ho1) may be derived from Prochlorococcus marinus.

[0135] Furthermore, the glucose dehydrogenase (gdhA) may be derived from Priestia megaterium. Furthermore, the biliverdin reductase (bvr) may be derived from Mycobacterium paraintracellulare.

[0136] Furthermore, the phycocyanin ferredoxin oxidoreductase (pcyA) may be derived from Micromonas pusilla.

[0137] In a sixth aspect, the present invention provides the following A or B or C:

[0138] A is any of the following methods:

[0139] A1) A method for constructing a recombinant Escherichia coli with enhanced 5-ALA production, comprising the following steps: performing the transformation according to 1) to 26) of the first aspect or 1) to 23) and 26) of the first aspect.

[0140] A2) A method for constructing a recombinant Escherichia coli with increased hemoglobin production, comprising the steps of: performing the transformation according to the transformations 1)-23), 26), and 27)-35) of the second aspect;

[0141] A3) A method for constructing a recombinant Escherichia coli with increased biliverdin production, comprising the following steps: performing the transformation according to 1)-23), 26), 27)-34), and 36) of the third aspect;

[0142] A4) A method for constructing a recombinant Escherichia coli with increased bilirubin production, comprising the steps of: performing the transformation according to 1)-23), 26), 27)-34), 36) and 37) of the fourth aspect;

[0143] A5) A method for constructing a recombinant Escherichia coli with increased phycocyanin production, comprising the steps of: performing the transformation according to 1)-23), 26), 27)-34), 36) and 38) of the fifth aspect;

[0144] A6) A method for producing or increasing 5-ALA production, comprising the following steps:

[0145] The recombinant Escherichia coli described in the first aspect is cultured using glucose as a carbon source, and the cells are collected; the cells are then cultured in a D-glucose transformation medium to obtain 5-ALA;

[0146] A7) A method for producing or increasing hemoglobin production, comprising the steps of:

[0147] Cultivating the recombinant Escherichia coli described in the second aspect using glucose as a carbon source and collecting the cells; then culturing the cells in a D-glucose transformation medium to obtain hemoglobin;

[0148] A8) A method for producing or increasing biliverdin production, comprising the steps of:

[0149] Cultivating the recombinant Escherichia coli described in the third aspect using glycerol as a carbon source and collecting the cells; then culturing the cells in a D-glucose transformation medium to obtain biliverdin;

[0150] A9) A method for producing or increasing bilirubin production, comprising the steps of:

[0151] Cultivating the recombinant Escherichia coli described in the fourth aspect using glycerol as a carbon source and collecting the cells; then culturing the cells in a D-glucose conversion medium to obtain bilirubin;

[0152] A10) A method for producing or increasing phycocyanin yield, comprising the following steps:

[0153] Cultivating the recombinant Escherichia coli described in the fifth aspect using glycerol as a carbon source and collecting the cells; then culturing the cells in a D-glucose transformation medium to obtain phycocyanin;

[0154] B is any of the following applications:

[0155] B1) Use of the recombinant Escherichia coli according to any one of aspects 1 to 5 in the preparation of a product for producing or increasing 5-ALA, heme, biliverdin, bilirubin or phycocyanin;

[0156] B2) Use of the recombinant Escherichia coli of the first aspect in producing or increasing 5-ALA production;

[0157] B3) Use of the recombinant Escherichia coli according to the second aspect in producing or increasing heme production;

[0158] B4) Use of the recombinant Escherichia coli according to the third aspect in producing or increasing biliverdin production;

[0159] B5) Use of the recombinant Escherichia coli according to the fourth aspect in producing or increasing bilirubin production;

[0160] B6) Use of the recombinant Escherichia coli according to the fifth aspect in producing or increasing the yield of phycocyanin;

[0161] C is any of the following products:

[0162] C1) A product for producing or increasing 5-ALA production, wherein the active ingredient is the recombinant Escherichia coli described in the first aspect;

[0163] C2) a product for producing or increasing heme production, wherein the active ingredient is the recombinant Escherichia coli described in the second aspect;

[0164] C3) A product for producing or increasing biliverdin production, wherein the active ingredient is the recombinant Escherichia coli described in the third aspect;

[0165] C4) a product for producing or increasing bilirubin production, the active ingredient of which is the recombinant Escherichia coli described in the fourth aspect;

[0166] C5) A product for producing or increasing the yield of phycocyanin, wherein the active ingredient is the recombinant Escherichia coli described in the fifth aspect.

[0167] The above-mentioned product may be a bacterial agent containing the recombinant bacteria and / or a culture of the recombinant bacteria.

[0168] The term "culture" refers to any liquid or solid product (i.e., fermentation product) containing a microbial population after artificial inoculation and cultivation. This refers to a product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of the microorganism or contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process.

[0169] Furthermore, the growth medium using glycerol and glucose as carbon sources contains one or both of glucose and glycerol.

[0170] In a specific embodiment of the present invention, the growth medium may include:

[0171] 1) Glucose growth medium composition and final concentrations are as follows: Na2HPO4: 25 mM, KH2PO4: 25 mM, NH4Cl: 50 mM, Na2SO4: 5 mM, MgSO4: 2 mM, glucose: 2% (g / 100 mL), yeast extract: 0.5% (g / 100 mL), trace elements: 50 μM FeCl3, 20 μM CaCl2, 10 μM MnCl2, 10 μM ZnSO4, 2 μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3, and H3BO3;

[0172] 2) Glycerol growth medium composition and final concentrations are as follows: Na2HPO4: 25 mM, KH2PO4: 25 mM, NH4Cl: 50 mM, Na2SO4: 5 mM, MgSO4: 2 mM, glycerol: 2% (g / 100 mL), yeast extract: 0.5% (g / 100 mL), trace elements: 50 μM FeCl3, 20 μM CaCl2, 10 μM MnCl2, 10 μM ZnSO4, 2 μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3, and H3BO3.

[0173] The D-glucose conversion medium can be as follows, dissolving the following components with water to reach the corresponding final concentrations: Na2HPO4: 25mM, KH2PO4: 25mM, MgCl2: 50mM, D-glucose: 20% (g / 100mL), NH4Cl: 0.5% (g / 100mL).

[0174] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the phosphotransferase G subunit (ptsG) is ACZ37321.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding the ptsG is Gene ID: GU134316.1 (2009.11.25).

[0175] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the glucokinase (glk) is CAD6007722.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: LR883050.1 (2020.10.30).

[0176] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the galactose repressor (galR) is CAD6004914.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: LR883050.1 (2020.10.30).

[0177] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the glucose transport cofactor (Zglf) is AAA27691.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding the glucose transport cofactor (Zglf) is M60615.1 (1993.4.26).

[0178] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the lipid A biosynthesis myristoyltransferase (lpxM) is AAC74925.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 945143 (2023.4.14).

[0179] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the arabinose transporter (araE) is CAD6004880.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 947341 (2023.4.14).

[0180] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the NAD(P) transhydrogenase (hereinafter referred to as sthA) is CAD6022916.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948461 (2023.4.14).

[0181] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the phosphate acetyltransferase (pta) is NP_416800.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the encoding gene is Gene ID: 946778 (2023.4.14).

[0182] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the acetyl-CoA synthetase (hereinafter referred to as acs) is CAD6022819.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948572 (2023.4.14).

[0183] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the transcriptional regulatory factor (arcA) is NP_418818.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948874 (2023.4.14).

[0184] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the pantothenate kinase (coaA) is NP_418405.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 948479 (2023.4.14).

[0185] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the phosphoglycerate dehydrogenase (serA) is NP_417388.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 945258 (2023.4.14).

[0186] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the cysteine / O-acetylserine efflux protein (eamA) is NP_416050.4, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946081 (2023.4.14).

[0187] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the threonine / homoserine efflux protein (rhtA) is NP_415334.1, and the NCBI reference sequence number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 947045 (2023.4.14).

[0188] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the lactate dehydrogenase (ldhA) is NP_415898.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946315 (2023.4.14).

[0189] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the pyruvate oxidase (hereinafter referred to as poxB) is CAD6018048.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946132 (2023.4.14).

[0190] In the present invention, the NCBI Reference Sequence number (NCBI Reference Sequence) of the phosphoenolpyruvate carboxylase (ppc) is NP_418391.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948457 (2023.4.14). In the present invention, the NCBI Reference Sequence number (NCBI Reference Sequence) of the isocitrate lyase inhibitor (iclR) is AAC76988.2, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948524 (2023.4.14).

[0191] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the succinate dehydrogenase A subunit (sdhA) is NP_415251.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 945402 (2023.4.14).

[0192] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the citrate synthase (gltA) is NP_415248.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 945323 (2024.8.6).

[0193] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the homoserine acetyltransferase (BsmetA) is WP_003218242.1, and the NCBI reference sequence number (NCBI Reference Sequence) of the encoding gene thereof is Gene ID: 64303965 (2023.11.20).

[0194] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the acetylhomoserine lyase (BsyjcI) is YP_007251742.1, and the NCBI reference sequence number (NCBI Reference Sequence) of the encoding gene is Gene ID: 14352972 (2013.2.14).

[0195] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the tetrahydrodipyridine N-acetyltransferase (BsdapH) is NP_389301.2, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 939193 (2021.4.3).

[0196] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the N-acetyldiaminopimelate aminotransferase (BspatA) is YP_007533352.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the encoding gene thereof is Gene ID: 14769020 (2015.2.3).

[0197] In the present invention, the NCBI Reference Sequence number (NCBI Reference Sequence) of the N-acetyldiaminopimelate deacetylase (BsdapL) is YP_007533371.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 14769040 (2015.2.3). In the present invention, the NCBI Reference Sequence number (NCBI Reference Sequence) of the glycine dehydrogenase (GYDH) is NP_217296.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 888493 (2017.12.14).

[0198] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the isocitrate dehydrogenase (icd) is NP_415654.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 945702 (2024.8.24).

[0199] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the 5-aminolevulinic acid synthase (hemA) is ADE85192.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 31490330 (2023.10.06).

[0200] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the porphyrinogen peroxidase (yfeX) is NP_416926.4, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946913 (2023.4.14).

[0201] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the porphobilinogen synthase (hemB) is NP_414903.4, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 945017 (2023.4.14).

[0202] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the hydroxymethylcholine synthase (hemC) is YP_026260.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 947759 (2023.4.14).

[0203] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the uroporphyrinogen decarboxylase (hemE) is NP_418425.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948497 (2023.4.14).

[0204] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the coproporphyrinogen oxidase (hemF) is NP_416931.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946908 (2023.4.14).

[0205] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the protoporphyrinogen oxidase (hemG) is NP_418292.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948331 (2023.4.14).

[0206] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the ferrochelatase (hemH) is WP_014605551.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 57267780 (2023.9.29).

[0207] In the present invention, the NCBI Reference Sequence Numbers (NCBI Reference Sequence) of the heme efflux protein (CcmABC) are UZR21256.1, UZR21256.1, and UZR21258.1, and the NCBI Reference Sequence Numbers (NCBI Reference Sequence) of the genes encoding them are Gene ID: CP111004.1:1444223-1446284 (2022.11.21). In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the heme oxygenase (ho1) is WP_011821074.1, and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding it is Gene ID: 60201361 (2023.4.10).

[0208] In the present invention, the NCBI Reference Sequence number of the glucose dehydrogenase (gdhA) is CAA30931.1, and the NCBI Reference Sequence number of the gene encoding it is GeneBank: X12370.1 (221-1006) (2008.10.23). In the present invention, the NCBI Reference Sequence number of the biliverdin reductase (bvr) is WP_008256343.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 66745074 (2024.6.6).

[0209] In the present invention, the NCBI reference sequence number (NCBI Reference Sequence) of the phycocyanin ferredoxin oxidoreductase (pcyA) is YP_214684.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 3294530 (2023.1.7).

[0210] In the above, the Escherichia coli is Escherichia coli BW25113.

[0211] In summary, the recombinant bacteria of the present invention are based on Escherichia coli, in which ptsG, galR, lpxM, pta, poxB, arcA, ldhA, sdhA, iclR, metA, and dapD are deleted; glk, Zglf, acs, ppc, BsmetA, BsyjcI, BsdapH, BspatA, BscdapL are introduced; the expression of araE, sthA, coaA, serA, and gltA is enhanced; the expression of icd is weakened; the expression of eamA and rhtA is enhanced; GYDH and hemA are introduced; this recombinant strain synthesizes 5-ALA using D-glucose as a raw material, and genes encoding heme and various derivatives thereof are introduced based on 5-ALA, thereby obtaining a recombinant bacteria that produces heme and various derivatives thereof.

[0212] The specific recombinant bacteria include the following aspects for the transformation of the recipient bacteria: a1), a2), a3), a4), a5), a6), a7), a8) and a9):

[0213] a1) Replace the gene encoding the phosphotransferase subunit G, ptsG, with the constitutive promoter P 119 The gene encoding glucokinase (glucokinase) glk (HA101) was replaced with the constitutive promoter P CPA1 and encoding glucose transport cofactors (galactose: + The Zglf gene encoding the symporter of lipid A was knocked out (HA102), and the lpxM gene encoding the lipid A biosynthesis myristoyltransferase was knocked out (HA103).

[0214] a2) Replace the gene encoding phosphate acetyltransferase (pta) with the constitutive promoter P CPA1The genes encoding acetyl-CoA synthetase, acs (HA106), arcA (HA108), ldhA (HA109), and poxB (HA107) were knocked out, and the expression of pantothenate kinase, serA (HA111), araE (HA104), and NAD(P) transhydrogenase (sthA) were enhanced.

[0215] a3) Replace the gene iclR encoding the isocitrate lyase repressor with the constitutive promoter P 119 The ppc gene encoding phosphoenolpyruvate carboxylase (HA115) was knocked out, the succinate dehydrogenase subunit A gene (sdhA) was knocked out (HA112), the expression of the citrate synthase gltA gene was enhanced (HA113), the expression of the isocitrate dehydrogenase icd gene was weakened (HA114), and the metA gene encoding homoserine succinyltransferase was replaced with the constitutive promoter P CPA1 The gene encoding homoserine acetyltransferase BsmetA and the gene encoding acetylhomoserine lyase BsyjcI (HA116) were replaced with the constitutive promoter P CPA1 and the gene encoding tetrahydrodipyridine N-acetyltransferase BsdapH, N-acetyldiaminopimelate aminotransferase BspatA, and N-acetyldiaminopimelate deacetylase BscdapL (HA117).

[0216] a4) Enhancing the expression of the cysteine / O-acetylserine efflux protein (eamA) gene (HA118) and the threonine / homoserine efflux protein (rhtA) gene (HA119).

[0217] a5) Glycine dehydrogenase GYDH and 5-aminolevulinic acid synthase hemA (HA121) were introduced into biological cells, and the genes encoding cysteine / O-acetylserine efflux protein eamA (HA122), threonine / homoserine efflux protein rhtA (HA123), and porphyrinogen peroxidase yfeX were knocked out (HA124), and the expression of genes encoding porphobilinogen synthase hemB (HA125), hydroxymethylbilane synthase hemC (HA126), uroporphyrinogen decarboxylase hemE (HA127), coproporphyrinogen III oxidase hemF (HA128), and protoporphyrinogen oxidase hemG (HA129) were enhanced. The ferrochelatase hemH is introduced into biological cells.

[0218] a6) The gene encoding the heme efflux protein (ABC transporter) CcmABC (HA130) was introduced.

[0219] a7) The gene ho1 encoding heme oxygenase and the gene gdhA (HA132) encoding glucose dehydrogenase were introduced.

[0220] a8) The gene encoding biliverdin reductase bvr (HA133) was introduced.

[0221] a9) The gene encoding phycocyanobilin ferredoxin-oxidoreductase, pcyA (HA134), was introduced.

[0222] In the methods described herein, method a1) improves the efficiency of substrate glucose entering cells and eliminates endotoxin production. Method a2) eliminates competitive metabolic bypasses, reduces carbon flow losses, and improves the metabolic efficiency of substrate glucose. Method a3) achieves a balanced supply of glycine and succinyl-CoA precursors. Method a4) provides microorganisms with the ability to synthesize 5-ALA from D-glucose. Method a5) provides microorganisms with the ability to synthesize heme from D-glucose. Method a6) provides microorganisms with the ability to synthesize heme from D-glucose outside the cell. Method a7) provides microorganisms with the ability to synthesize biliverdin from D-glucose. Method a8) provides microorganisms with the ability to synthesize bilirubin from D-glucose. Method a9) provides microorganisms with the ability to synthesize phycocyanin from D-glucose.

[0223] The present invention achieves the following beneficial technical effects: The recombinant strain provided by the present invention can achieve microbial growth using glucose or glycerol as a carbon source, and can also utilize mixed carbon sources containing these carbon sources. After the microorganisms have grown to a certain biomass, 5-ALA, heme, and various derivatives thereof are synthesized using D-glucose as a raw material. Furthermore, a corresponding technical route for synthesizing 5-ALA, heme, and various derivatives thereof has been established.

[0224] Preservation Instructions

[0225] (1) Strain number: HA130

[0226] Species name: Escherichia coli

[0227] Latin name: Escherichia coli

[0228] Depository: General Microbiology Center of China Culture Collection Administration

[0229] Abbreviation of depository institution: CGMCC

[0230] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0231] Deposit date: October 17, 2024

[0232] CGMCC registration number: CGMCC No.32252

[0233] Classification name: Escherichia coli

[0234] (2) Strain number: HA133

[0235] Species name: Escherichia coli

[0236] Latin name: Escherichia coli

[0237] Depository: General Microbiology Center of China Culture Collection Administration

[0238] Abbreviation of depository institution: CGMCC

[0239] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0240] Deposit date: October 17, 2024

[0241] CGMCC registration number: CGMCC No.32251

[0242] Classification name: Escherichia coli

[0243] (3) Strain number: HA134

[0244] Species name: Escherichia coli

[0245] Latin name: Escherichia coli

[0246] Depository: General Microbiology Center of China Culture Collection Administration

[0247] Abbreviation of depository institution: CGMCC

[0248] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0249] Deposit date: October 17, 2024

[0250] CGMCC registration number: CGMCC No.32253

[0251] Classification name: Escherichia coli DETAILED DESCRIPTION

[0252] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0253] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0254] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0255] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0256] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0257] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0258] Escherichia coli BW25113 (CGSC#:7636), plasmids pKD46 (CGSC#:7739), pKD4 (CGSC#:7682), and pCP20 (CGSC#:7629) were purchased from the Yale Escherichia coli Collection (CGSC).

[0259] In the following examples, unless otherwise specified, substitutions refer to complete substitutions and knockouts refer to complete knockouts.

[0260] The primers and partial fragment sequences in the following examples are shown in Table 1.

[0261] Table 1 shows the sequences of the primers

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] The nucleotide sequence of the RBS in the following examples is 5'-AGGAGGAATTAAC-3'.

[0269] The nucleotide sequence of the linker in the following examples is 5′-GAGGGAAAAGGCGGCAGTGGCTCTGGTAGTGAGGGCGGCGCGAAAGGTGGAGGCTCT-3′.

[0270] Example 1. Construction of recombinant Escherichia coli HA121 and production of 5-ALA

[0271] 1. This example prepared a basic bacterium HA121 that can use glucose or glycerol as a carbon source for bacterial growth and can convert D-glucose into 5-ALA. The preparation method of this strain is obtained according to the following steps (1)-(10):

[0272] (1) Replace the gene encoding phosphotransferase G subunit (hereinafter referred to as ptsG) with the gene encoding glucokinase (glk)

[0273] Starting from Escherichia coli BW25113, the gene encoding the phosphotransferase G subunit (ptsG) in the strain was replaced with the constitutive promoter P 119 and a gene encoding glucokinase (glk) to obtain recombinant Escherichia coli HA101, the specific steps are as follows:

[0274] (1-a) Preparation of the targeting fragment ptsGup-kan-P 119 -glk-ptsGdown

[0275] The DNA fragments synthesized by GeneScript (GenScript) were as follows: from 5' to 3', they consisted of the following: a 70 bp homology arm fragment upstream of the ptsG gene (denoted as ptsGup, see Table 1), an frt-kan-frt screening marker fragment (SEQ ID No. 3), a P 119 The promoter fragment (SEQ ID No. 2), glk gene, TrrnB terminator fragment (SEQ ID No. 4), and 70 bp downstream fragment of ptsG gene (denoted as ptsGdown, see Table 1) were amplified by PCR using ptsG-1 / ptsG-2 as primers and the gene-synthesized DNA fragment as template to obtain the targeting fragment ptsGup-kan-P 119 -glk-ptsGdown.

[0276] (1-b) Preparation of host bacteria containing pKD46 plasmid

[0277] The pKD46 plasmid (from the Yale Genetic Collection of E. coli, CGSC) was transformed into E. coli BW25113 using the calcium chloride method. After overnight culture at 30°C on LB plates containing ampicillin, colonies were selected to obtain recombinant E. coli BW25113 / pKD46 containing the pKD46 plasmid. After induction with arabinose, BW25113 / pKD46 expressed the three recombinant proteins of λ phage, conferring homologous recombination capability. Competent BW25113 / pKD46 cells were then prepared by washing with 10% glycerol.

[0278] (1-c) Homologous recombination

[0279] The targeting fragment ptsGup-kan-P prepared in (1-a) 119 -glk-ptsGdown was electroporated into the BW25113 / pKD46 competent cells prepared in (1-b) and incubated overnight at 37°C on an LB plate containing kanamycin (50 μg / ml). A clone was selected to extract genomic DNA and amplified by PCR using ptsG-2 / Kan-R primers. A positive clone was identified by amplifying a target band of approximately 1000 bp. The positive clone was named HA101-kan. Sequencing analysis results showed that the genome of HA101-kan contained the ptsGup-kan-P 119 -glk-ptsGdown fragment.

[0280] HA101-kan was cultured at 42°C overnight to eliminate the temperature-sensitive plasmid pKD46.

[0281] (1-d) Elimination of resistance

[0282] The pCP20 plasmid (from the Yale Genetic Collection of Escherichia coli (CGSC)) was transformed into HA101-kan using the calcium chloride method. The cells were cultured overnight at 30°C on LB plates containing ampicillin. Kanamycin resistance was eliminated using the FLP recombinase on the pCP20 plasmid. The temperature-sensitive plasmid pCP20 was eliminated by overnight culture at 42°C. Single clones were identified by PCR amplification using primers ptsG-2 / ptsG-3. A positive result for the approximately 1300-bp target band was obtained, indicating recombinant strain HA101.

[0283] The NCBI Reference Sequence number of the phosphotransferase G subunit (ptsG) is ACZ37321.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: GU134316.1 (2009.11.25).

[0284] The NCBI Reference Sequence number (NCBI Reference Sequence) of glucokinase (glk) is CAD6007722.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: LR883050.1 (2020.10.30).

[0285] (2) Replace the gene encoding the galactose repressor (galR) with the gene encoding the glucose transport cofactor (Zglf)

[0286] Starting from the recombinant strain HA101, the gene encoding the galactose repressor (galR) in the strain was replaced with the constitutive promoter P CPA1 and a gene encoding a glucose transport cofactor (Zglf) from Zymomonas mobilis to obtain recombinant Escherichia coli HA102. The specific steps are as follows:

[0287] (2-a) Preparation of the targeting fragment galRup-kan-P CPA1 -Zglf-galRdown

[0288] The DNA fragments synthesized by GeneScript (GenScript) are as follows: from 5' to 3', they consist of the following: a 70 bp homology arm fragment upstream of the galR gene (denoted as galRup, see Table 1), an frt-kan-frt selection marker fragment (SEQ ID No. 3), a P CPA1 The promoter fragment (SEQ ID No. 1), Zglf gene, TrrnB terminator fragment (SEQ ID No. 4), and 70 bp downstream fragment of galR gene (denoted as galRdown, see Table 1) were amplified by PCR using galR-1 / galR-2 as primers and the gene-synthesized DNA fragment as template to obtain the targeting fragment galRup-kan-P CPA1 -Zglf-galRdown.

[0289] (2-b) The subsequent operations refer to (1) (steps 1-b to 1-d) to obtain the constitutive promoter P CPA1 The recombinant strain HA102 in which the galR gene is replaced by the Zglf gene is described. The difference is that the host strain BW25113 is replaced by HA101, and the primers ptsG-1 / ptsG-2 / ptsG-3 are replaced by galR-1 / galR-2 / galR-3. In step (2-c), PCR amplification and identification are performed using galR-2 / Kan-R as primers, and the amplification of the target band of approximately 1000 bp is considered positive. In step (2-d), PCR amplification and identification are performed using galR-2 / galR-3 as primers, and the amplification of the target band of approximately 2000 bp is considered positive.

[0290] The NCBI reference sequence number (NCBI Reference Sequence) of the galactose repressor (galR) is CAD6004914.1, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: LR883050.1 (2020.10.30).

[0291] The NCBI Reference Sequence Number of the glucose transport cofactor (Zglf) is AAA27691.1, and the NCBI Reference Sequence Number of the gene encoding it is M60615.1 (1993.4.26).

[0292] (3) Knockout of the lipid A biosynthesis myristoyltransferase (lpxM) gene

[0293] Starting from the recombinant strain HA102, the coding region of the myristoyltransferase gene lpxM of the recombinant strain HA102 was deleted to obtain the recombinant strain HA103. The specific steps are as follows:

[0294] (3-a) Preparation of the targeting fragment lpxMup-kan-lpxMdown

[0295] PCR amplification was performed using lpxM-1 / lpxM-2 as primers and the frt-kan-frt screening marker fragment as a template to obtain the targeting fragment lpxMup-kan-lpxMdown.

[0296] (3-b) Subsequent operations were performed as described in (1) (steps 1-b to 1-d) to obtain the recombinant strain HA103 with the lpxM gene knocked out. The differences were that the host strain BW25113 was replaced with HA102, and the primers ptsG-1 / ptsG-2 / ptsG-3 were replaced with lpxM-1 / lpxM-2 / lpxM-3.

[0297] In step (3-c), PCR amplification and identification were performed using lpxM-2 / Kan-R as primers, and the amplification of a target band of about 1000 bp was considered positive;

[0298] In step (3-d), PCR amplification and identification were performed using lpxM-2 / lpxM-3 as primers, and the amplified target band of about 200 bp was considered positive.

[0299] The NCBI Reference Sequence number of lipid A biosynthesis myristoyltransferase (lpxM) is AAC74925.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 945143 (2023.4.14).

[0300] (4) Enhanced expression of the arabinose transporter (araE) gene by promoter replacement

[0301] Starting from the recombinant strain HA103, the promoter of the arabinose transporter araE gene (GenBank: CP032667.1 (901033-901192) 08-OCT-2018) in the strain was replaced with the Escherichia coli constitutive promoter P CPA1 (SEQ ID No. 1), and recombinant Escherichia coli HA104 was obtained by the following steps:

[0302] (4-a) Preparation of the targeting fragment araEup-kan-P CPA1 -araEdown

[0303] The DNA fragments synthesized (GenScript) were as follows: from 5' to 3', they consisted of the following: a 70 bp homology arm fragment upstream of the araE gene (denoted as araEup, see Table 1), an frt-kan-frt screening marker fragment, a P CPA1 The promoter fragment (SEQ ID No. 1) and the first 70 bp fragment of the araE gene (denoted as sequence araEdown, see Table 1) were amplified by PCR using araE-1 / araE-2 as primers and the gene-synthesized DNA fragment as a template to obtain the targeting fragment araEup-kan-P CPA1 -araEdown.

[0304] (4-b) The subsequent operations refer to (1) (steps 1-b to 1-d) to obtain the constitutive promoter P CPA1 The recombinant strain HA104 with the araE promoter replaced. The differences are that the host strain BW25113 was replaced by HA103, and the primers ptsG-1 / ptsG-2 / ptsG-3 were replaced by araE-1 / araE-2 / araE-3.

[0305] In step (4-c), PCR amplification and identification were performed using araE-2 / Kan-R as primers, and the amplification of a target band of about 1000 bp was considered positive;

[0306] In step (4-d), PCR amplification and identification were performed using araE-2 / araE-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0307] The NCBI Reference Sequence number (NCBI Reference Sequence) of the arabinose transporter (araE) is CAD6004880.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 947341 (2023.4.14).

[0308] (5) Enhanced expression of the NAD(P) transhydrogenase (sthA) gene by promoter replacement

[0309] Referring to the step (4), on the basis of the recombinant bacteria HA104, the promoter of the NAD(P) transhydrogenase (sthA) gene (GenBank: NC_000913.3(4160791-4160860)09-MAR-2022) was replaced with the Escherichia coli constitutive promoter P 119 , and recombinant E. coli HA105 was obtained. The difference is that the gene synthesis fragments are as follows: from 5' to 3', they are composed of the following: sthAup, frt-kan-frt screening marker fragment, P 119 The promoter fragment (SEQ ID No. 2), sthAdown, the primer combination araE-1 / araE-2 / araE-3 were replaced with sthA-1 / sthA-2 / sthA-3 in sequence, and the host bacteria HA103 was replaced with HA104.

[0310] The sequence of the upstream homology arm of sthA is sthAup, see Table 1; the sequence of the downstream homology arm of sthA is sthAdown, see Table 1;

[0311] PCR amplification was performed using sthA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0312] PCR amplification and identification were performed using sthA-2 / sthA-3 as primers, and the amplified target band of about 300 bp was considered positive.

[0313] The NCBI Reference Sequence number (NCBI Reference Sequence) of NAD(P) transhydrogenase (sthA) is CAD6022916.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948461 (2023.4.14).

[0314] (6) Replace the gene encoding phosphate acetyltransferase (pta) with the gene encoding acetyl-CoA synthetase (acs)

[0315] Starting from the recombinant strain HA105, the gene encoding phosphate acetyltransferase (pta) in the strain was replaced with the constitutive promoter P CPA1 and the gene encoding acetyl-CoA synthetase (acs) to obtain recombinant Escherichia coli HA106. The specific steps are as follows:

[0316] (6-a) Preparation of the targeting fragment pta up-kan-P CPA1 -acs-pta down

[0317] The DNA fragments synthesized by GeneScript (GenScript) are as follows: from 5' to 3', they consist of the following: a 70 bp homology arm fragment upstream of the pta gene (denoted as pta up, see Table 1), an frt-kan-frt selection marker fragment (SEQ ID No. 3), a P CPA1 The promoter fragment (SEQ ID No. 1), acs gene, TrrnB terminator fragment (SEQ ID No. 4), and 70 bp fragment downstream of the pta gene (denoted as pta down, see Table 1) were amplified by PCR using pta-1 / pta-2 as primers and the gene-synthesized DNA fragment as a template to obtain the targeting fragment pta up-kan-P CPA1 -acs-pta down.

[0318] (6-b) The subsequent operations refer to (1) (steps 1-b to 1-d) to obtain the constitutive promoter P CPA1 The recombinant strain HA106 in which the pta gene was replaced by the acs gene was constructed. The differences were that the host strain BW25113 was replaced by HA105, and the primers ptsG-1 / ptsG-2 / ptsG-3 were replaced by pta-1 / pta-2 / pta-3.

[0319] PCR amplification was performed using pta-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0320] PCR amplification and identification were performed using pta-2 / pta-3 as primers, and the amplified target band of about 2400 bp was considered positive.

[0321] The NCBI Reference Sequence number (NCBI Reference Sequence) of phosphate acetyltransferase (pta) is NP_416800.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946778 (2023.4.14).

[0322] The NCBI Reference Sequence number of acetyl-CoA synthetase (acs) is CAD6022819.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 948572 (2023.4.14).

[0323] (7) Knockout of the pyruvate oxidase (poxB) gene, the transcriptional regulatory factor (arcA) gene, and the lactate dehydrogenase (ldhA) gene

[0324] Referring to the steps of (3), on the basis of HA106, the pyruvate oxidase gene poxB, the transcriptional regulatory factor gene arcA, and the lactate dehydrogenase gene ldhA were knocked out in sequence to obtain recombinant bacteria HA107, HA108, and HA109.

[0325] The differences are that the host strain HA102 was replaced with HA106, HA107, and HA108, respectively, and the primer combinations lpxM-1 / lpxM-2 / lpxM-3 were replaced with poxB-1 / poxB-2 / poxB-3, arcA-1 / arcA-2 / arcA-3, and ldhA-1 / ldhA-2 / ldhA-3, respectively. The resulting targeting fragments were poxBup-kan-poxBdown, arcAup-kan-arcAdown, and ldhAup-kan-ldhAdown, respectively. The targeting fragments differed from those in 3-a) only in the primers used. The relevant primer sequences are shown in Table 1.

[0326] PCR amplification was performed using poxB-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0327] PCR amplification was performed using poxB-2 / poxB-3 as primers, and the amplified target band of about 200 bp was considered positive.

[0328] PCR amplification was performed using arcA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0329] PCR amplification and identification were performed using arcA-2 / arcA-3 as primers, and the amplified target band of about 200 bp was considered positive.

[0330] PCR amplification was performed using ldhA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0331] PCR amplification and identification were performed using ldhA-2 / ldhA-3 as primers, and the amplified target band of about 200 bp was considered positive.

[0332] The NCBI Reference Sequence number (NCBI Reference Sequence) of pyruvate oxidase (poxB) is CAD6018048.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946132 (2023.4.14).

[0333] The NCBI Reference Sequence number of the transcriptional regulatory factor (arcA) is NP_418818.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 948874 (2023.4.14).

[0334] The NCBI Reference Sequence number of lactate dehydrogenase (ldhA) is NP_415898.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 946315 (2023.4.14).

[0335] (8) Enhanced expression of pantothenate kinase (coaA) and phosphoglycerate dehydrogenase (serA) genes by promoter replacement

[0336] Referring to the step (4), on the basis of the recombinant bacteria HA109, the promoter of the pantothenate kinase (coaA) gene (GenBank: NC_000913.3 (4175027-4175186) 09-MAR-2022) and the promoter of the phosphoglycerate dehydrogenase (serA) gene (GenBank: NC_000913.3 (3058411-3058570) 09-MAR-2022) were replaced with the Escherichia coli constitutive promoter P CPA1 , and obtained recombinant Escherichia coli HA110 and HA111.

[0337] The differences are that the transformed strain HA103 was replaced by HA109 and HA110 in sequence, the homologous arm sequences in the synthesized gene fragments were replaced by coaAup / coaAdown and serAup / serAdown in sequence, and the primer araE-1 / araE-2 / araE-3 combination was replaced by coaA-1 / coaA-2 / coaA-3 and serA-1 / serA-2 / serA-3 in sequence.

[0338] The coaAup, coaAdown, serAup, serAdown sequences and related primer sequences are shown in Table 1.

[0339] PCR amplification was performed using coaA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0340] PCR amplification and identification were performed using coaA-2 / coaA-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0341] PCR amplification was performed using serA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0342] PCR amplification and identification were performed using serA-2 / serA-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0343] The NCBI Reference Sequence number (NCBI Reference Sequence) of pantothenate kinase (coaA) is NP_418405.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948479 (2023.4.14).

[0344] The NCBI Reference Sequence number of phosphoglycerate dehydrogenase (serA) is NP_417388.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 945258 (2023.4.14).

[0345] (9) Knockout of the succinate dehydrogenase A subunit (sdhA) gene

[0346] Referring to the steps in (3), the coding region of the sdhA gene for the succinate dehydrogenase A subunit was deleted from HA111 to obtain the recombinant strain HA112. The differences were that the host strain HA102 was replaced with HA111, and the primer combination lpxM-1 / lpxM-2 / lpxM-3 was replaced with sdhA-1 / sdhA-2 / sdh-3. The target fragment obtained in the experiment was sdhAup-kan-sdhAdown. The relevant primer sequences are shown in Table 1.

[0347] PCR amplification was performed using sdhA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0348] PCR amplification was performed using sdhA-2 / sdhA-3 as primers, and the amplified target band of about 200 bp was considered positive.

[0349] The NCBI Reference Sequence number (NCBI Reference Sequence) of succinate dehydrogenase A subunit (sdhA) is NP_415251.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 945402 (2023.4.14).

[0350] (10) Enhanced expression of the citrate synthase (gltA) gene by promoter replacement

[0351] Referring to the steps of (5), on the basis of the recombinant bacteria HA112, the promoter of the citrate synthase (gltA) gene (GenBank: NC_000913.3 (754469-754868) 09-MAR-2022) was replaced with the Escherichia coli constitutive promoter P 119 , and obtained recombinant Escherichia coli HA113.

[0352] The differences are that the transformed strain HA104 was replaced with HA112, the homology arm sequences in the synthesized gene fragment were replaced with gltAup / gltAdown, and the primer combination sthA-1 / sthA-2 / sthA-3 was replaced with gltA-1 / gltA-2 / gltA-3. The gltAup and gltAdown sequences and the corresponding primer sequences are shown in Table 1.

[0353] PCR amplification was performed using gltA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0354] PCR amplification and identification were performed using gltA-2 / gltA-3 as primers, and the amplified target band of about 300 bp was considered positive.

[0355] The NCBI Reference Sequence number (NCBI Reference Sequence) of the citrate synthase (gltA) is NP_415248.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 945323 (2024.8.6).

[0356] (11) Weakening the expression of the isocitrate dehydrogenase (icd) gene by promoter replacement

[0357] Referring to the step (4), on the basis of the recombinant bacteria HA113, the promoter of the isocitrate dehydrogenase (icd) gene (GenBank: NC_000913.3 (1194961-1195122) 09-MAR-2022) was replaced with the Escherichia coli constitutive promoter P 113 , and recombinant E. coli HA114 was obtained. The difference is that the gene synthesis fragments are as follows: from 5' to 3', they are composed of the following: icdup, frt-kan-frt screening marker fragment, P 113 The promoter fragment (SEQ ID No. 5), icddown, and primer combination araE-1 / araE-2 / araE-3 were replaced with icd-1 / icd-2 / icd-3 in sequence, and the host strain HA103 was replaced with HA113.

[0358] The sequences of Icdup, icddown, and related primers are shown in Table 1 ;

[0359] PCR amplification was performed using icd-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0360] PCR amplification and identification were performed using icd-2 / icd-3 as primers, and the amplified target band of about 300 bp was considered positive.

[0361] The NCBI Reference Sequence number (NCBI Reference Sequence) of isocitrate dehydrogenase (icd) is NP_415654.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 945702 (2024.8.24).

[0362] (12) Replace the gene encoding isocitrate lyase inhibitor (iclR) with the gene encoding phosphoenolpyruvate carboxylase (ppc)

[0363] Starting from the recombinant strain HA114, the gene encoding the isocitrate lyase inhibitor (iclR) in the strain was replaced with the constitutive promoter P 119 and a gene encoding phosphoenolpyruvate carboxylase (ppc) to obtain recombinant Escherichia coli HA115. The specific steps are as follows:

[0364] (12-a) Preparation of the targeting fragment iclR up-kan-P 119 -ppc-iclR down

[0365] The DNA fragments synthesized (GenScript) were as follows: from 5' to 3', they consisted of the following: a 70 bp homology arm fragment upstream of the iclR gene (denoted as iclR up, see Table 1), an frt-kan-frt screening marker fragment (SEQ ID No. 3), a P 119 The promoter fragment (SEQ ID No. 2), ppc gene, TrrnB terminator fragment (SEQ ID No. 4), and 70 bp fragment downstream of the iclR gene (denoted as iclR down, see Table 1) were amplified by PCR using iclR-1 / iclR-2 as primers and the gene-synthesized DNA fragment as a template to obtain the targeting fragment iclR up-kan-P 119 -ppc-iclR down.

[0366] (12-b) The subsequent operations refer to (1) (steps 1-b to 1-d) to obtain the constitutive promoter P 119 The recombinant strain HA115 in which the iclR gene was replaced by the ppc gene was different in that the host strain BW25113 was replaced by HA114 and the primers ptsG-1 / ptsG-2 / ptsG-3 were replaced by iclR-1 / iclR-2 / iclR-3.

[0367] PCR amplification was performed using iclR-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0368] PCR amplification was performed using iclR-2 / iclR-3 as primers, and the amplified target band of about 3100 bp was considered positive.

[0369] Among them, the NCBI reference sequence number (NCBI Reference Sequence) of the isocitrate lyase inhibitor (iclR) is AAC76988.2, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948524 (2023.4.14).

[0370] The NCBI Reference Sequence number of phosphoenolpyruvate carboxylase (ppc) is NP_418391.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 948457 (2023.4.14).

[0371] (13) The gene encoding homoserine succinyltransferase (metA) was replaced with genes encoding homoserine acetyltransferase (BsmetA) and acetylhomoserine lyase (BsyjcI)

[0372] Starting from the recombinant strain HA115, the gene encoding homoserine succinyltransferase (metA) in the strain was replaced with the constitutive promoter P CPA1 and genes encoding homoserine acetyltransferase (BsmetA) and acetylhomoserine lyase (BsyjcI) to obtain recombinant Escherichia coli HA116. The specific steps are as follows:

[0373] (13-a) Preparation of targeting fragment metA up-kan-P CPA1 -BsmetA-BsyjcI-metA down

[0374] The DNA fragments synthesized by GeneScript (GenScript) were as follows: from 5' to 3', they consisted of the following: a 70 bp homology arm fragment upstream of the metA gene (denoted as metA up, see Table 1), an frt-kan-frt selection marker fragment (SEQ ID No. 3), a P CPA1 The promoter fragment (SEQ ID No. 1), BsmetA gene, rbs regulatory sequence, BsyjcI gene, TrrnB terminator fragment (SEQ ID No. 4), and 70 bp fragment downstream of metA gene (denoted as metA down, see Table 1) were amplified by PCR using metA-1 / metA-2 as primers and gene-synthesized DNA fragment as template to obtain the targeting fragment metA up-kan-P CPA1 -BsmetA-BsyjcI-metA down.

[0375] (13-b) The subsequent operations refer to (1) (steps 1-b to 1-d) to obtain the constitutive promoter P CPA1 The recombinant strain HA116 in which the metA gene was replaced by the BsmetA and BsyjcI genes was different in that the host strain BW25113 was replaced by HA115 and the primers ptsG-1 / ptsG-2 / ptsG-3 were replaced by metA-1 / metA-2 / metA-3.

[0376] PCR amplification was performed using metA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0377] PCR amplification and identification were performed using metA-2 / metA-3 as primers, and the amplified target band of about 2500 bp was considered positive.

[0378] The NCBI Reference Sequence number (NCBI Reference Sequence) of homoserine succinyltransferase (metA) is NP_418437.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948513 (2023.4.14).

[0379] The NCBI Reference Sequence number of homoserine acetyltransferase (BsmetA) is WP_003218242.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 64303965 (2023.11.20).

[0380] The NCBI Reference Sequence number of acetylhomoserine lyase (BsyjcI) is YP_007251742.1, and the NCBI Reference Sequence number of the gene encoding the acetylhomoserine lyase (BsyjcI) is Gene ID: 14352972 (2013.2.14).

[0381] (14) The gene encoding tetrahydrodipyridine N-succinate transferase (dapD) was replaced with genes encoding tetrahydrodipyridine N-acetyltransferase (BsdapH), N-acetyldiaminopimelate aminotransferase (BspatA), and N-acetyldiaminopimelate deacetylase (BsdapL).

[0382] Starting from the recombinant strain HA116, the gene encoding tetrahydrodipyridyl N-succinate transferase (dapD) in the strain was replaced with the constitutive promoter P CPA1 and genes encoding tetrahydrodipyridine N-acetyltransferase (BsdapH), N-acetyldiaminopimelate aminotransferase (BspatA), and N-acetyldiaminopimelate deacetylase (BsdapL) to obtain recombinant Escherichia coli HA117. The specific steps are as follows:

[0383] (14-a) Preparation of the targeting fragment dapD up-kan-P CPA1 -BsdapH-BspatA-BsdapL-dapD down

[0384] The DNA fragments synthesized (GenScript) were as follows: from 5' to 3', they consisted of the following: a 70 bp homology arm fragment upstream of the dapD gene (denoted as dapD up, see Table 1), an frt-kan-frt selection marker fragment (SEQ ID No. 3), a PCPA1 The promoter fragment (SEQ ID No. 1), BsdapH gene, rbs regulatory sequence, BspatA gene, rbs regulatory sequence, BsdapL gene, TrrnB terminator fragment (SEQ ID No. 4), and 70 bp downstream fragment of dapD gene (denoted as dapD down, see Table 1) were amplified by PCR using dapD-1 / dapD-2 as primers and the gene-synthesized DNA fragment as template to obtain the targeting fragment dapD up-kan-P CPA1 -BsdapH-BspatA-BsdapL-dapD down.

[0385] (14-b) The subsequent operations refer to (1) (steps 1-b to 1-d) to obtain the constitutive promoter P CPA1 The recombinant strain HA116 in which the metA gene was replaced by the BsdapH, BspatA, and BsdapL genes was obtained. The difference was that the host strain BW25113 was replaced by HA116, and the primers ptsG-1 / ptsG-2 / ptsG-3 were replaced by dapD-1 / dapD-2 / dapD-3.

[0386] PCR amplification was performed using dapD-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0387] PCR amplification and identification were performed using dapD-2 / dapD-3 as primers, and the amplified target band of about 3400 bp was considered positive.

[0388] The NCBI Reference Sequence number (NCBI Reference Sequence) of tetrahydrodipyridine N-succinate transferase (dapD) is NP_414708.1, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 944862 (2024.8.6).

[0389] The NCBI reference sequence number (NCBI Reference Sequence) of tetrahydrodipyridine N-acetyltransferase (BsdapH) is NP_389301.2, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 939193 (2021.4.3).

[0390] The NCBI Reference Sequence number of N-acetyldiaminopimelate aminotransferase (BspatA) is YP_007533352.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 14769020 (2015.2.3).

[0391] The NCBI Reference Sequence number of N-acetyldiaminopimelate deacetylase (BsdapL) is YP_007533371.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 14769040 (2015.2.3).

[0392] (15) Enhanced expression of cysteine / O-acetylserine efflux protein (eamA) and threonine / homoserine efflux protein (rhtA) genes by promoter replacement

[0393] Referring to the step (4), on the basis of the recombinant bacteria HA117, the promoters of cysteine / O-acetylserine efflux protein (eamA) (GenBank: NC_000913.3 (1621138-1621200) 09-MAR-2022) and threonine / homoserine efflux protein (rhtA) (GenBank: NC_000913.3 (850098-850147) 09-MAR-2022) were replaced with the Escherichia coli constitutive promoter P CPA1 , and obtained recombinant Escherichia coli HA118 and HA119.

[0394] The differences are that the transformed strain HA103 was replaced by HA117 and HA118 in sequence, the homologous arm sequences in the synthesized gene fragments were replaced by eamAup / eamAdown and rhtAup / rhtAdown in sequence, and the primer combination araE-1 / araE-2 / araE-3 was replaced by eamA-1 / eamA-2 / eamA-3 and rhtA-1 / rhtA-2 / rhtA-3 in sequence.

[0395] The sequences of eamAup, eamAdown, rhtAup, rhtAdown and related primers are shown in Table 1.

[0396] PCR amplification was performed using eamA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0397] PCR amplification and identification were performed using eamA-2 / eamA-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0398] PCR amplification was performed using rhtA-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0399] PCR amplification was performed using rhtA-2 / rhtA-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0400] The NCBI reference sequence number (NCBI Reference Sequence) of the cysteine / O-acetylserine efflux protein (eamA) is NP_416050.4, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946081 (2023.4.14).

[0401] The NCBI Reference Sequence number of threonine / homoserine efflux protein (rhtA) is NP_415334.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 947045 (2023.4.14).

[0402] (16) The genes encoding glycine dehydrogenase (GYDH) and 5-aminolevulinic acid synthase (hemA) were introduced into the recombinant bacterium HA119.

[0403] Starting from the recombinant bacterium HA119, the expression plasmid pSB1s-GYDH-hemA containing the GYDH and hemA genes was introduced into HA119 to obtain HA121. The specific steps are as follows:

[0404] (16-a) Preparation of pSB1s-GYDH-hemA Plasmid

[0405] A DNA fragment containing GYDH and hemA was synthesized by Qingke Biotechnology. From the 5' to the 3' end, it consists of the GYDH gene, RBS, and hemA gene. Using the synthesized sequence as a template and primers GYDH-F and hemA-R, the GYDH-hemA gene fragment was amplified by PCR using high-fidelity TransStart Fast Pfu DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd., catalog number AP221). The target fragment was recovered by agarose gel electrophoresis.

[0406] Then, using primers pSB1s-01F and pSB1s-01R and plasmid pSB1s (SEQ ID No. 6) as a template, PCR amplified the pSB1s fragment and recovered the large vector fragment pSB1s. The GYDH-hemA fragment was ligated with the pSB1s fragment using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to 100 kilobases. Nat. Methods. 2009; 6(5): 343-345) to generate the recombinant expression vector pSB1s-GYDH-hemA. Escherichia coli DH5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product catalog number CD201) were transformed using the CaCl2 method. The cells were evenly plated on LB plates containing streptomycin and cultured overnight at 37°C. The clones were selected and the primers araBADP-F / TrrnB-R were used to identify the clones that could amplify the target fragment and sequenced. The positive clones were selected to extract the plasmids and the obtained positive plasmids were named pSB1s-GYDH-hemA.

[0407] (16-b) Plasmid transformation

[0408] The pSB1s-GYDH-hemA plasmid was transformed into recombinant strain HA111 using the calcium chloride method. After overnight culture at 37°C on LB plates containing streptomycin, clones were selected. Positive clones were identified using primers araBADP-F / TrrnB-R and stored frozen at -80°C. The recombinant strain was designated HA120A.

[0409] The pSB1s-GYDH-hemA plasmid was transformed into recombinant strain HA117 using the calcium chloride method. After overnight culture at 37°C on LB plates containing streptomycin, clones were selected. Positive clones were identified using primers araBADP-F / TrrnB-R and stored frozen at -80°C. The recombinant strain was designated HA120B.

[0410] The pSB1s-GYDH-hemA plasmid was transformed into recombinant strain HA119 using the calcium chloride method. After overnight culture at 37°C on LB plates containing streptomycin, clones were selected. Positive clones were identified using primers araBADP-F / TrrnB-R and stored frozen at -80°C. The recombinant strain was designated HA121.

[0411] The NCBI Reference Sequence number of glycine dehydrogenase (GYDH) is NP_195027.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 829438 (2024.8.22).

[0412] The NCBI Reference Sequence number of 5-aminolevulinic acid synthase (hemA) is ADE85192.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 31490330 (2023.10.06).

[0413] 2. Cultivation of recombinant bacteria HA121 using glucose as a carbon source and production of 5-ALA using glucose as a substrate

[0414] 1. Glucose Growth Medium Composition

[0415] The growth medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0416] Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, glucose: 3% (g / 100mL), yeast powder: 0.5% (g / 100mL), trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3;

[0417] 2. Transformation medium components

[0418] The transformation medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0419] Na2HPO4: 25 mM, KH2PO4: 25 mM, MgCl2: 50 mM, D-glucose: 10% (g / 100 mL), NH4Cl: 1% (g / 100 mL);

[0420] 3. Bacterial Culture and Enzyme Induction

[0421] The wild-type strain BW25113, recombinant bacteria HA120A, recombinant bacteria HA120B and recombinant bacteria HA121 were cultured overnight at a 1% inoculum rate in a shake flask containing 200 ml of glucose growth medium (wherein the growth medium of HA120A, HA120B and HA121 contained streptomycin at a final concentration of 100 mg / L) and cultured at 30°C for 3-4 h until the OD 600nm After the concentration of arabinose reached 0.6-0.8, 0.2 g / L arabinose was added, and the culture was continued at 30°C for 12 h. The bacteria were collected by centrifugation at 8000 rpm for 10 min.

[0422] Whole-cell catalysis of 4.5-ALA

[0423] Resuspend the bacteria collected in the above 3 in a beaker containing 10 ml of transformation medium, with a final concentration of 50 O.D., and obtain the transformation liquid for reaction. The reaction process is regularly detected using pH test paper, and adjusted with 0.1M NaOH and HCl solution to maintain the pH at 7.0. After reacting at 37°C for 12 hours, the transformation liquid is centrifuged and the supernatant is filtered. Take 200μl of a certain dilution of the supernatant, add 100μl 1M pH4.6 sodium acetate and 50μl acetylacetone, mix well and boil in water bath for 15 minutes, cool to room temperature, take 200μl of the above reaction liquid, add 200μl Ehrlich reagent (Shanghai Yuanye Biotechnology Co., Ltd., product number R20690), and after standing at room temperature for 20 minutes, use a microplate reader to detect the 554nm absorption value. The standard curve is Y=0.0167X+0.1047(R 2 =0.9991, Y=absorbance at 554 nm / X=5-ALA concentration in the reaction system (mg / L). The 5-ALA yield in the conversion solution was obtained by conversion.

[0424] The results showed that the 5-ALA production of the wild-type strain BW25113 was 0, the 5-ALA production of the recombinant strain HA120A was 12 g / L, the 5-ALA production of the recombinant strain HA120B was 33.5 g / L, and the average 5-ALA production of the engineered strain HA121 was 42.1 g / L.

[0425] Example 2: Construction of recombinant Escherichia coli HA130 and production of heme

[0426] 1. This example prepared a basic bacterium HA130 that can use glucose as a carbon source for bacterial growth and convert D-glucose into heme. The preparation method of this strain is as follows:

[0427] (1) Knockout of the cysteine / O-acetylserine efflux protein (eamA) gene, the threonine / homoserine efflux protein (rhtA) gene, and the porphyrinogen peroxidase (yfeX) gene

[0428] Starting from the recombinant bacterium HA117, the eamA gene, rhtA gene, and yfeX gene of the recombinant bacterium HA117 were knocked out in sequence to obtain recombinant bacteria HA122, HA123, and HA124. The specific steps are described in (3) of Example 1. The primer combinations used were eamA-4 / eamA-5 / eamA-3, rhtA-4 / rhtA-5 / rhtA-3, and yfeX-1 / yfeX-2 / yfeX-3, respectively.

[0429] The targeting fragment eamAup-kan-eamAdown was amplified by PCR using primers eamA-4 / eamA-5 and the frt-kan-frt selection marker as a template. PCR amplification using primers eamA-3 / Kan-R identified a positive target band of approximately 1000 bp. PCR amplification using primers eamA-3 / eamA-2 identified a positive target band of approximately 200 bp.

[0430] The target fragment was amplified by PCR using rhtA-4 / rhtA-5 primers and the frt-kan-frt screening marker fragment as a template, resulting in the target fragment rhtAup-kan-rhtAdown. PCR amplification using rhtA-3 / Kan-R primers identified the target band as approximately 1000 bp, while PCR amplification using rhtA-3 / rhtA-2 primers identified the target band as approximately 200 bp.

[0431] The targeting fragment yfeXup-kan-yfeXdown was amplified by PCR using primers yfeX-1 / yfeX-2 and the frt-kan-frt screening marker fragment as a template. PCR amplification using primers yfeX-3 / Kan-R amplified a target band of approximately 1000 bp, indicating a positive result. PCR amplification using primers yfeX-3 / yfeX-2 amplified a target band of approximately 200 bp, indicating a positive result.

[0432] Among them, the NCBI reference sequence number (NCBI Reference Sequence) of cysteine / O-acetylserine efflux protein (eamA) is NP_416050.4, and the NCBI reference sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946081 (2023.4.14).

[0433] The NCBI Reference Sequence number of threonine / homoserine efflux protein (rhtA) is NP_415334.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 947045 (2023.4.14).

[0434] The NCBI Reference Sequence number of porphyrinogen peroxidase (yfeX) is NP_416926.4, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 946913 (2023.4.14).

[0435] (2) Enhanced expression of genes encoding porphobilinogen synthase (hemB), hydroxymethylbilinogen synthase (hemC), uroporphyrinogen decarboxylase (hemE), coproporphyrinogen oxidase (hemF), and protoporphyrinogen oxidase (hemG) by promoter replacement

[0436] Starting from the recombinant bacterium HA124, the promoter of the gene encoding porphobilinogen synthase (hemB) (GenBank: NC_000913.3 (389728-389777) 09-MAR-2022), the promoter of the gene encoding hydroxymethylbilinogen synthase (hemC) (GenBank: NC_000913.3 (3990767-3990773) 09-MAR-2022), and the promoter of the gene encoding uroporphyrinogen decarboxylase (hemE) (GenBank: NC_000913.3 (3990767-3990773) 09-MAR-2022) in the strain were cloned. 0913.3(4197681-4197705)09-MAR-2022), the promoter of the gene encoding coproporphyrinogen oxidase (hemF) (GenBank: NC_000913.3(2552952-2553224)09-MAR-2022), and the promoter of the gene encoding protoporphyrinogen oxidase (hemG) (GenBank: NC_000913.3(4031147-4031160)09-MAR-2022) were replaced with the Escherichia coli constitutive promoter P 119, recombinant Escherichia coli HA125, HA126, HA127, HA128 and HA129 were obtained. The specific steps were as described in Example 1 (4). The difference was that the homology arm sequences araEup / araEdown in the gene synthesis fragment were replaced by hemBup / hemBdwon, hemCup / hemCdown, hemEup / hemEdown, hemFup / hemFdown and hemGup / hemGdown, respectively. CPA1 The promoter fragment (SEQ ID No. 1) was replaced by P 119 Promoter fragment (SEQ ID No. 2), the primer combination araE-1 / araE-2 / araE-3 was replaced by hemB-1 / hemB-2 / hemB-3, hemC-1 / hemC-2 / hemC-3, hemE-1 / hemE-2 / hemE-3, hemF-1 / hemF-2 / hemF-3 and hemG-1 / hemG-2 / hemG-3, respectively.

[0437] The sequences of hemBup, hemBdwon, hemCup, hemCdown, hemEup, hemEdown, hemFup, hemFdown, hemGup, and hemGdown are shown in Table 1.

[0438] PCR amplification was performed using hemB-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0439] PCR amplification and identification were performed using hemB-2 / hemB-3 as primers, and the amplified target band of about 300 bp was considered positive.

[0440] PCR amplification was performed using hemC-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0441] PCR amplification and identification were performed using hemC-2 / hemC-3 as primers, and the amplified target band of about 300 bp was considered positive.

[0442] PCR amplification was performed using hemE-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0443] PCR amplification and identification were performed using hemE-2 / hemE-3 as primers, and the amplified target band of about 300 bp was considered positive.

[0444] PCR amplification was performed using hemF-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0445] PCR amplification and identification were performed using hemF-2 / hemF-3 as primers, and the amplified target band of about 300 bp was considered positive.

[0446] PCR amplification was performed using hemG-2 / Kan-R as primers, and the amplified target band of about 1000 bp was considered positive;

[0447] PCR amplification and identification were performed using hemG-2 / hemG-3 as primers, and the amplified target band of about 300 bp was considered positive.

[0448] Among them, the NCBI Reference Sequence number (NCBI Reference Sequence) of porphobilinogen synthase (hemB) is NP_414903.4, and the NCBI Reference Sequence number (NCBI Reference Sequence) of its encoding gene is Gene ID: 945017 (2023.4.14).

[0449] The NCBI Reference Sequence number of hydroxymethylcholine synthase (hemC) is YP_026260.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 947759 (2023.4.14).

[0450] The NCBI Reference Sequence number of uroporphyrinogen decarboxylase (hemE) is NP_418425.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 948497 (2023.4.14).

[0451] The NCBI Reference Sequence number of coproporphyrinogen oxidase (hemF) is NP_416931.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 946908 (2023.4.14).

[0452] The NCBI Reference Sequence number of protoporphyrinogen oxidase (hemG) is NP_418292.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 948331 (2023.4.14).

[0453] (3) Introducing the gene encoding ferrochelatase (hemH) into the recombinant bacterium HA129

[0454] Starting from the recombinant bacteria HA129, the expression plasmid pYB1k-hemH-CcmABC containing the hemH gene was introduced into HA129 to obtain HA130. The specific steps are as follows:

[0455] (3-a) Preparation of pYB1k-hemH-CcmABC plasmid

[0456] DNA fragments containing hemH and CcmABC were synthesized by Qingke Biotechnology. Using the synthesized sequences as templates and primers hemH-F / hemH-R and CcmABC-F / CcmABC-R, respectively, high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221) was used to amplify the hemH and CcmABC gene fragments by PCR. The target fragments were recovered by agarose gel electrophoresis.

[0457] Then, using primers pYB1k-01F and pYB1k-01R and plasmid pYB1k (SEQ ID No. 7) as a template, PCR amplification of the pYB1k fragment was performed, and the large vector fragment pYB1k was recovered. The hemH and CcmABC fragments were ligated with the pYB1k fragment using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to selpxMeral hundred kilobases. Nat. Methods. 2009; 6(5): 343-345) to generate the recombinant expression vector pYB1k-hemH-CcmABC. Escherichia coli DH5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number CD201) were transformed using the CaCl2 method. The cells were evenly plated on LB plates containing streptomycin and cultured overnight at 37°C. The clones were selected and the primers araBADP-F / TrrnB-R were used to identify the clones that could amplify the target fragment and sequenced. The positive clones were selected to extract the plasmids and the obtained positive plasmids were named pYB1k-hemH-CcmABC.

[0458] (3-b) Plasmid transformation

[0459] The pYB1k-hemH-CcmABC plasmid and the pSB1s-GYDH-hemA plasmid obtained in Example 1-(10) were co-transformed into recombinant strain HA129 using the calcium chloride transformation method. After overnight culture at 37°C on LB plates containing streptomycin and kanamycin, clones were selected. Positive clones were identified using primers araBADP-F / TrrnB-R and stored frozen at -80°C. The strain was designated Escherichia coli HA130.

[0460] Escherichia coli HA130 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on October 17, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 32252, and the classification name is Escherichia coli.

[0461] The NCBI Reference Sequence number of ferrochelatase (hemH) is WP_014605551.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 57267780 (2023.9.29).

[0462] The NCBI Reference Sequence numbers of the heme efflux proteins (CcmABC) are UZR21256.1, UZR21256.1, and UZR21258.1, and the NCBI Reference Sequence numbers of the genes encoding them are Gene ID: CP111004.1:1444223-1446284 (2022.11.21). In the present invention, the NCBI Reference Sequence number of the heme oxygenase (ho1) is WP_011821074.1, and the NCBI Reference Sequence number of the gene encoding it is Gene ID: 60201361 (2023.4.10).

[0463] 2. Cultivation of recombinant bacteria HA130 using glucose as a carbon source and preparation of heme using glucose as a substrate

[0464] 1. Glucose Growth Medium Composition

[0465] The growth medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0466] Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, glucose: 3% (g / 100mL), yeast powder: 0.5% (g / 100mL), trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3;

[0467] 2. Transformation medium components

[0468] The transformation medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0469] Na2HPO4: 25 mM, KH2PO4: 25 mM, MgCl2: 50 mM, D-glucose: 10% (g / 100 mL), NH4Cl: 0.5% (g / 100 mL);

[0470] 3. Bacterial Culture and Enzyme Induction

[0471] The wild-type strain BW25113, recombinant strain HA129 and engineered strain HA130 were cultured overnight at a 1% inoculum rate in a shake flask containing 200 ml of glucose growth medium (wherein the HA130 growth medium contained streptomycin at a final concentration of 100 mg / L and kanamycin at a final concentration of 100 mg / L) and cultured at 30°C for 3-4 h until the OD 600nm After the concentration of arabinose reached 0.6-0.8, 0.2 g / L arabinose was added, and the culture was continued at 30°C for 12 h. The bacteria were collected by centrifugation at 8000 rpm for 10 min.

[0472] 4. Whole-cell catalysis of heme

[0473] The collected bacteria were resuspended in beakers containing 10 ml of conversion solution A. or B. The final concentration of bacteria was 50O.D., and the pH was maintained at 7.0 during the reaction. The two conversion solutions were: A. Conversion medium; B. Conversion medium with 5-ALA added at a final concentration of 1%. After reacting at 37°C for 24 hours, the supernatant was centrifuged and filtered. The hemoglobin content was detected by HPLC. HPLC used a waters C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was methanol / phosphoric acid aqueous solution (40 / 60), the flow rate was 0.5 mL / min; the column temperature was 35°C; the detection wavelength was 405 nm. The hemoglobin standard was purchased from Aladdin (item number: H104216). The retention time of the hemoglobin standard was 16.9 min. The standard curve is Y = 95.471X + 114.855 (R 2=0.9993, Y=peak area / X=heme concentration in the loading bottle). The heme yield in the conversion solution was calculated. The results are shown in Table 2.

[0474] Table 2 is a comparison of hemoglobin production

[0475] conversion fluid BW25113(g / L) HA129(g / L) HA130(g / L) A. 0 0 3.05 B. 0 0 3.28

[0476] The results showed that the engineered strain HA130 could effectively synthesize hemoglobin.

[0477] Example 3. Construction of recombinant Escherichia coli HA132 and production of biliverdin

[0478] 1. This example prepared a basic bacterium HA132 that can use glycerol as a carbon source for bacterial growth and can convert D-glucose into biliverdin. The preparation method of the strain is as follows:

[0479] The expression plasmid containing the heme oxygenase (ho1) encoding gene was introduced into the recombinant bacteria HA129 to obtain recombinant Escherichia coli HA131 and HA132. The specific steps are as follows:

[0480] (1) Preparation of pYB1k1-HemH-ho1-GDH and pYB1k2-HemH-ho1-GDH plasmids

[0481] HemH-RBS-ho1-RBS-gdhA (composed sequentially of the HemH gene, RBS, ho1 gene, RBS, and gdhA) and HemH-linker-ho1-RBS-gdhA fragments (composed sequentially of the HemH gene (excluding TAA), linker, ho1 gene, RBS, and gdhA) were synthesized. Using the synthesized sequences as templates and hemH-F / ho1-R as primers, respectively, high-fidelity TransStart Fast Pfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221) was used to amplify the HH1 (corresponding to HemH-RBS-ho1-RBS-gdhA) and HH2 (corresponding to HemH-linker-ho1-RBS-gdhA) fragments. The desired fragments were recovered by agarose gel electrophoresis. Then, using primers pYB1k-01F and pYB1k-01R and plasmid pYB1k as a template, PCR amplification of the pYB1k fragment was performed, and the large vector fragment pYB1k was recovered. The HH1 and HH2 fragments were ligated with the pYB1k fragment using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods. 2009; 6(5):343-345), respectively, to generate the recombinant expression vectors pYB1k1-HemH-ho1-gdhA and pYB1k2-HemH-ho1-ghdA. Escherichia coli DH5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number CD201) were transformed using the CaCl2 method. The cells were evenly plated on LB plates containing kanamycin and cultured overnight at 37°C. The clones were selected and the primers araBADP-F / TrrnB-R were used to identify the clones that could amplify the target fragments and sequenced. The positive clones were selected to extract the plasmids. The obtained positive plasmids were named pYB1k1-HemH-ho1-gdhA and pYB1k2-HemH-ho1-gdhA.

[0482] (10-b) Plasmid transformation

[0483] The pYB1k1-HemH-ho1-ghdA and pYB1k2-HemH-ho1-gdhA plasmids were co-transformed with the pSB1s-GYDH-hemA obtained in Example 1-(10) into the recombinant strain HA129 by the calcium chloride transformation method. After overnight culture at 37°C on LB plates containing streptomycin and kanamycin, clones were selected. Positive clones were identified using primers araBADP-F / TrrnB-R and frozen at -80°C. The strains were named Escherichia coli HA131 and HA132.

[0484] The NCBI Reference Sequence number of heme oxygenase (ho1) is WP_011821074.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 60201361 (2023.4.10).

[0485] The NCBI Reference Sequence number of glucose dehydrogenase (gdhA) is CAA30931.1, and the NCBI Reference Sequence number of the gene encoding it is GeneBank: X12370.1 (221-1006) (2008.10.23).

[0486] 2. Cultivation of recombinant bacteria HA131 and HA132 using glycerol as a carbon source and preparation of biliverdin using glucose as a substrate

[0487] 1. Glycerol Growth Medium Composition

[0488] The growth medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0489] Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, glycerol: 3% (g / 100mL), yeast powder: 0.5% (g / 100mL), trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3;

[0490] 2. Transformation medium components

[0491] The transformation medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0492] Na2HPO4: 25 mM, KH2PO4: 25 mM, MgCl2: 50 mM, D-glucose: 10% (g / 100 mL), NH4Cl: 0.5% (g / 100 mL);

[0493] 3. Bacterial Culture and Enzyme Induction

[0494] The wild-type strain BW25113, recombinant strain HA131 and engineered strain HA132 were cultured overnight at a 1% inoculum rate in a shake flask containing 200 ml of glycerol growth medium (wherein the HA126 growth medium contained streptomycin at a final concentration of 100 mg / L, and the HA127 growth medium contained streptomycin and kanamycin at a final concentration of 100 mg / L), and cultured at 30°C for 3-4 h until the OD 600nm After the concentration of arabinose reached 0.6-0.8, 0.2 g / L arabinose was added, and the culture was continued at 30°C for 12 h. The bacteria were collected by centrifugation at 8000 rpm for 10 min.

[0495] 4. Whole-cell catalysis of biliverdin

[0496] The collected bacteria were resuspended in a beaker containing 10 ml of conversion solution. The final concentration of the bacteria was 50 O.D. and the pH was maintained at 7.0 during the reaction. After reacting at 37 ° C for 24 hours, the bacteria were centrifuged. The bacteria were resuspended in 0.1 M potassium phosphate buffer (pH 7.0), ultrasonically disrupted and centrifuged to obtain the precipitate. After dichloromethane extraction, the precipitate was centrifuged to obtain the organic phase, and the biliverdin content was detected by HPLC. HPLC used a waters C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was DMSO / acetonitrile / 0.5 M ammonium phosphate buffer (5:7:7), the flow rate was 0.5 mL / min; the column temperature was 30 ° C; the detection wavelength was 380 nm. The biliverdin standard was purchased from Aladdin (item number: B344750). The retention time of the biliverdin standard was 13.2 min. The standard curve was Y = 33924.9X-33924.5 (R 2 =0.999Y=peak area / X=biliverdin concentration in the loading bottle (mg / L). Calculate the biliverdin yield in the conversion solution.

[0497] The results showed that the biliverdin production of the wild-type strain BW25113 was 0, the biliverdin production of the recombinant strain HA131 was 1.17 g / L, and the average biliverdin production of the engineered strain HA132 was 2.05 g / L.

[0498] Example 4. Construction of recombinant Escherichia coli HA133 and production of bilirubin

[0499] 1. This example prepared a recombinant strain HA133 that can use glycerol as a carbon source for bacterial growth and can convert D-glucose into bilirubin. The preparation method of the strain is as follows:

[0500] The expression plasmid pYB1k-hemH-ho1-bvr-gdhA containing the coding genes for heme oxygenase (ho1) and biliverdin reductase (bvr) and the pSB1s-GYDH-hemA obtained in Example 1-(10) were introduced into the recombinant bacteria HA129 to obtain recombinant Escherichia coli HA133. The specific steps are as follows:

[0501] (1) Preparation of pYB1k-hemH-ho1-bvr-gdhA plasmid

[0502] The bvr gene fragment was synthesized by Qingke Biotechnology. Using the synthetic bvr gene fragment as a template and bvr-01F / bvr-01R as primers, the bv fragment was amplified using high-fidelity TransStart FastPfu DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd., catalog number AP221). The target fragment was recovered by agarose gel electrophoresis. Then, using primers pYB1k-02F and pYB1k-02R and plasmid pYB1k2-hemH-ho1-gdhA as a template, PCR amplification of the pYB1k2-hemH-ho1::gdhA fragment was performed. The large vector fragment, pYB1k2-hemH-ho1::gdhA, was recovered. The bvr fragment was ligated with the pYB1k2-hemH-ho1::gdhA fragment using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods. 2009; 6(5): 343-345) to generate the recombinant expression vector pYB1k-hemH-ho1-bvr-gdhA. Competent Escherichia coli DH5α cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product catalog number CD201) were transformed using the CaCl2 method. These cells were evenly plated on LB plates containing kanamycin and cultured overnight at 37°C. Clones were selected and identified using primers araBADP-F / TrrnB-R to amplify the target fragment. These clones were sequenced and the plasmids were extracted from positive clones. The resulting plasmid was named pYB1k-hemH-ho1-bvr-gdhA.

[0503] (10-b) Plasmid transformation

[0504] The pYB1k-hemH-ho1-bvr-gdhA plasmid and the pSB1s-GYDH-hemA obtained in Example 1-(10) were co-transformed into the recombinant strain HA129 by the calcium chloride transformation method. After overnight culture at 37°C on LB plates containing streptomycin and kanamycin, clones were selected. Positive clones were identified using primers araBADP-F / TrrnB-R and frozen at -80°C. The strain was named Escherichia coli HA133.

[0505] Escherichia coli HA133 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on October 17, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 32251, and the classification name is Escherichia coli.

[0506] The NCBI Reference Sequence number of biliverdin reductase (bvr) is WP_008256343.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 66745074 (2024.6.6).

[0507] 2. Cultivation of recombinant bacteria HA133 using glycerol as a carbon source and glucose as a substrate for bilirubin production

[0508] 1. Glycerol Growth Medium Composition

[0509] The growth medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0510] Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, glycerol: 3% (g / 100mL), yeast powder: 0.5% (g / 100mL), trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3;

[0511] 2. Transformation medium components

[0512] The transformation medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0513] Na2HPO4: 25 mM, KH2PO4: 25 mM, MgCl2: 50 mM, D-glucose: 10% (g / 100 mL), NH4Cl: 0.5% (g / 100 mL).

[0514] 3. Bacterial Culture and Enzyme Induction

[0515] The wild-type strain BW25113, recombinant strain HA129 and engineered strain HA133 were cultured overnight at a 1% inoculum rate in a shake flask containing 200 ml of glycerol growth medium (wherein the HA133 growth medium contained streptomycin and kanamycin at a final concentration of 100 mg / L) and cultured at 30°C for 3-4 h until the OD 600nm After the concentration of arabinose reached 0.6-0.8, 0.2 g / L arabinose was added, and the culture was continued at 30°C for 12 h. The bacteria were collected by centrifugation at 8000 rpm for 10 min.

[0516] 4. Whole-cell catalysis of bilirubin

[0517] Resuspend the collected cells in a beaker containing 10 ml of either transformation medium A or B, to a final concentration of 50 OD. Maintain the pH at 7.0 during the reaction. The two transformation mediums are: A. Transformation medium; B. Transformation medium supplemented with 1% 5-ALA, 0.1% hemin, and 0.02% Triton X-100. After incubation at 37°C for 24 hours, the cells were centrifuged. Resuspend the cells in 0.1 M potassium phosphate buffer (pH 7.0), ultrasonically disrupt, and centrifuge to obtain the precipitate. Extract the precipitate with dichloromethane, then centrifuge the organic phase, and analyze the bilirubin content by HPLC. The HPLC was performed using a Waters C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase consisted of DMSO / acetonitrile / 0.5 M ammonium phosphate buffer (5:7:7), at a flow rate of 0.5 mL / min; the column temperature was 30°C; and the detection wavelength was 453 nm. Bilirubin standard was purchased from Aladdin (Cat. No. B104211). The retention time of bilirubin standard was 20 min. The standard curve was Y = 10570.4X - 11660.7 (R 2 =0.993Y = peak area / X = bilirubin concentration in the loading bottle (mg / L). The bilirubin yield in the conversion solution was calculated. The results are shown in Table 3.

[0518] Table 3. Comparison of bilirubin production

[0519] conversion fluid BW25113(g / L) HA129(g / L) HA133(g / L) A. 0 0 2.11 B. 0 0 2.51

[0520] The results showed that the engineered strain HA133 could effectively synthesize bilirubin.

[0521] Example 5. Construction of recombinant Escherichia coli HA134 and production of phycocyanin

[0522] 1. This example prepared a basic strain HA134 that can use glycerol as a carbon source for bacterial growth and convert D-glucose into phycocyanin. The preparation method of this strain is as follows:

[0523] The expression plasmid pYB1k-hemH-ho1-pcyA-gdhA containing the coding genes for heme oxygenase (ho1) and phycocyanin ferredoxin oxidoreductase (pcyA) was introduced into the recombinant bacterium HA129 to obtain recombinant Escherichia coli HA134. The specific steps are as follows:

[0524] (1) Preparation of pYB1k-hemH-ho1-pcyA-gdhA plasmid

[0525] The pcyA gene fragment was synthesized by Qingke Biotechnology. Using the synthesized pcyA gene fragment as a template, pcyA-01F and pcyA-01R as primers, high-fidelity TransStart FastPfu DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd., catalog number AP221) was used to amplify the pcyA fragment. The target fragment was then recovered by agarose gel electrophoresis. Then, using primers pYB1k-02F and pYB1k-02R and plasmid pYB1k2-hemH-ho1 as a template, PCR amplification of the pYB1k2-hemH-ho1::gdhA fragment was performed. The large vector fragment, pYB1k2-hemH-ho1::gdhA, was recovered. The pcyA fragment was ligated with the pYB1k2-hemH-ho1::gdhA fragment using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods. 2009; 6(5): 343-345) to generate the recombinant expression vector pYB1k-HHP. Competent Escherichia coli DH5α cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product catalog number CD201) were transformed using the CaCl2 method. These cells were evenly plated on LB plates containing kanamycin and cultured overnight at 37°C. Clones were selected and identified using primers araBADP-F / TrrnB-R to amplify the target fragment. These clones were sequenced and the plasmids were extracted from positive clones. The resulting plasmid was named pYB1k-hemH-ho1-pcyA-gdhA.

[0526] (10-b) Plasmid transformation

[0527] The pYB1k-hemH-ho1-pcyA-gdhA plasmid and the pSB1s-GYDH-hemA obtained in Example 1-(10) were co-transformed into the recombinant strain HA129 by the calcium chloride transformation method. After overnight culture at 37°C on LB plates containing streptomycin and kanamycin, clones were selected. Positive clones were identified using primers araBADP-F / TrrnB-R and frozen at -80°C. The strain was named Escherichia coli HA134.

[0528] Escherichia coli HA134 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on October 17, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 32253, and the classification name is Escherichia coli.

[0529] The NCBI Reference Sequence number of phycocyanin ferredoxin oxidoreductase (pcyA) is YP_214684.1, and the NCBI Reference Sequence number of its encoding gene is Gene ID: 3294530 (2023.1.7).

[0530] 2. Cultivation of recombinant bacteria HA134 using glycerol as carbon source and glucose as substrate for phycocyanin production

[0531] 1. Glycerol Growth Medium Composition

[0532] The growth medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0533] Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, glycerol: 3% (g / 100mL), yeast powder: 0.5% (g / 100mL), trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3;

[0534] 2. Transformation medium components

[0535] The transformation medium components and final concentrations are as follows: Dissolve the following components in water to the corresponding final concentrations:

[0536] Na2HPO4: 25 mM, KH2PO4: 25 mM, MgCl2: 50 mM, D-glucose: 20% (g / 100 mL);

[0537] 3. Bacterial Culture and Enzyme Induction

[0538] The wild-type strain BW25113, recombinant strain HA129, and engineered strain HA134 were cultured overnight at a 1% inoculum rate in a shake flask containing 200 ml of glycerol growth medium (wherein the HA129 growth medium contained streptomycin at a final concentration of 100 mg / L, and the HA134 growth medium contained streptomycin and kanamycin at a final concentration of 100 mg / L), and cultured at 30°C for 3-4 h until the OD 600nm After the concentration of arabinose reached 0.6-0.8, 0.2 g / L arabinose was added, and the culture was continued at 30°C for 12 h. The bacteria were collected by centrifugation at 8000 rpm for 10 min.

[0539] 4. Whole-cell catalysis of phycocyanin

[0540] Resuspend the collected cells in a beaker containing 10 ml of either conversion medium A or B, respectively, to a final concentration of 50 OD. Maintain the pH at 7.0 during the reaction. The two conversion solutions are: A. Transformation medium; B. Transformation medium supplemented with 1% 5-ALA, 0.1% hemin, and 0.02% Triton X-100. After incubation at 37°C for 24 hours, the cells were harvested by centrifugation. Resuspend the cells in a small amount of methanol, extract with ethyl acetate, and centrifuge. The supernatant was analyzed by HPLC for phycocyanin content. The HPLC analysis was performed using a Waters C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase consisted of a pH 7.0 buffer solution containing 0.5 mol / L potassium dihydrogen phosphate and potassium hydrogen phosphate, at a flow rate of 0.3 mL / min; the column temperature was 78°C; and the detection wavelength was 687 nm. Phycocyanin standards were purchased from TCI (Cat. No. P2201). The retention time of the phycocyanin standard was 11 minutes. The standard curve is Y=86772.5X+23126.3(R 2 =0.998Y=peak area / X=phycocyanin concentration in the sample bottle (mg / L). The phycocyanin yield in the conversion solution was calculated. The results are shown in Table 4.

[0541] Table 4 is a comparison of phycocyanin production

[0542] conversion fluid BW25113(g / L) HA129(g / L) HA134(g / L) A. 0 0 1.82 B. 0 0 2.03

[0543] The results showed that the engineered strain HA134 could effectively synthesize phycocyanin.

[0544] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A recombinant Escherichia coli, which is obtained by carrying out the following transformations 1)-23) and 26) in Escherichia coli; 1) Knock out the gene encoding the phosphotransferase G subunit, ptsG; 2) Introducing the glucokinase coding gene glk and its expression regulatory factors; 3) Knock out the galactose repressor gene galR; 4) Introducing the gene encoding the glucose transport cofactor Zglf and its expression regulating factor; 5) Knockout the gene lpxM encoding myristoyltransferase in lipid A biosynthesis; 6) Enhance the expression of araE, the gene encoding the arabinose transporter; 7) Enhance the expression of sthA, the gene encoding NAD(P) transhydrogenase; 8) Knock out the gene encoding phosphate acetyltransferase, pta; 9) Introducing the acetyl-CoA synthetase encoding gene acs and its expression regulating factor; 10) Knock out the gene encoding pyruvate oxidase, poxB; 11) Knock out the gene encoding the transcriptional regulator arcA; 12) Knock out the gene encoding lactate dehydrogenase, ldhA; 13) Enhance the expression of coaA, the gene encoding pantothenate kinase; 14) Enhancing the expression of serA, the gene encoding phosphoglycerate dehydrogenase; 15) Knock out the gene encoding sdhA of succinate dehydrogenase A subunit; 16) Enhancing the expression of the gene gltA encoding citrate synthase; 17) Weakening the expression of the gene encoding isocitrate dehydrogenase, icd; 18) Knock out the gene encoding isocitrate lyase inhibitor iclR; 19) Introducing the ppc gene encoding phosphoenolpyruvate carboxylase and its expression regulating factor; 20) Knock out metA, the gene encoding homoserine succinyltransferase; 21) introducing the homoserine acetyltransferase encoding gene BsmetA and the acetylhomoserine lyase encoding gene BsyjcI and their expression regulating factors; 22) Knock out the dapD gene encoding tetrahydrobipyridine N-succinate transferase; 23) introducing the gene encoding tetrahydrobipyridine N-acetyltransferase BsdapH, the gene encoding N-acetyldiaminopimelate aminotransferase BspatA, and the gene encoding N-acetyldiaminopimelate deacetylase BscdapL and their expression level regulating factors; 26) The gene encoding glycine dehydrogenase GYDH and the gene encoding 5-aminolevulinic acid synthase hemA were introduced.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The modification also includes 24) enhancing the expression of the gene eamA encoding the cysteine / O-acetylserine efflux protein; 25) Enhance the expression of rhtA, the gene encoding threonine / homoserine efflux protein.

3. A recombinant Escherichia coli, wherein the transformations 1) to 23), 26) and the following transformations are carried out in Escherichia coli: 27) Knockout the gene eamA encoding cysteine / O-acetylserine efflux protein; 28) Knock out the gene rhtA encoding threonine / homoserine efflux protein; 29) Knock out the porphyrinogen peroxidase encoding gene yfeX; 30) Enhance the expression of hemB, the gene encoding porphobilinogen synthase; 31) enhancing the expression of the gene hemC encoding hydroxymethylcholine synthase; 32) enhancing the expression of hemE, the gene encoding uroporphyrinogen decarboxylase; 33) enhancing the expression of the gene hemF encoding coproporphyrinogen oxidase; 34) enhancing the expression of hemG, the gene encoding protoporphyrinogen oxidase; 35) Introduce the ferrochelatase encoding gene hemH and the heme efflux protein gene cluster CcmABC.

4. The recombinant Escherichia coli according to claim 3, characterized in that: The deposit number of the recombinant Escherichia coli is CGMCC No.32252.

5. A recombinant Escherichia coli, comprising the steps of carrying out the transformations 1) to 23), 26) and the following transformations in Escherichia coli: 27) Knockout the gene eamA encoding cysteine / O-acetylserine efflux protein; 28) Knock out the gene rhtA encoding threonine / homoserine efflux protein; 29) Knock out the porphyrinogen peroxidase encoding gene yfeX; 30) Enhance the expression of hemB, the gene encoding porphobilinogen synthase; 31) enhancing the expression of the gene hemC encoding hydroxymethylcholine synthase; 32) enhancing the expression of hemE, the gene encoding uroporphyrinogen decarboxylase; 33) enhancing the expression of the gene hemF encoding coproporphyrinogen oxidase; 34) enhancing the expression of hemG, the gene encoding protoporphyrinogen oxidase; 36) The gene encoding ferrochelatase, hemH, the gene encoding heme oxygenase, ho1, and the gene encoding glucose dehydrogenase, gdhA, were introduced.

6. A recombinant Escherichia coli, comprising the steps of carrying out the transformations 1) to 23), 26) and the following transformations in claim 1 in Escherichia coli: 27) Knockout the gene eamA encoding cysteine / O-acetylserine efflux protein; 28) Knock out the gene rhtA encoding threonine / homoserine efflux protein; 29) Knock out the porphyrinogen peroxidase encoding gene yfeX; 30) Enhance the expression of hemB, the gene encoding porphobilinogen synthase; 31) enhancing the expression of the gene hemC encoding hydroxymethylcholine synthase; 32) enhancing the expression of hemE, the gene encoding uroporphyrinogen decarboxylase; 33) enhancing the expression of the gene hemF encoding coproporphyrinogen oxidase; 34) enhancing the expression of hemG, the gene encoding protoporphyrinogen oxidase; 36) introducing the gene encoding ferrochelatase hemH, the gene encoding heme oxygenase ho1, and the gene encoding glucose dehydrogenase gdhA; 37) Introduce the gene encoding biliverdin reductase bvr.

7. The recombinant Escherichia coli according to claim 6, characterized in that: The deposit number of the recombinant Escherichia coli is CGMCC No.32251.

8. A recombinant Escherichia coli, comprising the following steps: 27) Knockout the gene eamA encoding cysteine / O-acetylserine efflux protein; 28) Knock out the gene rhtA encoding threonine / homoserine efflux protein; 29) Knock out the porphyrinogen peroxidase encoding gene yfeX; 30) Enhance the expression of hemB, the gene encoding porphobilinogen synthase; 31) enhancing the expression of the gene hemC encoding hydroxymethylcholine synthase; 32) enhancing the expression of hemE, the gene encoding uroporphyrinogen decarboxylase; 33) enhancing the expression of the gene hemF encoding coproporphyrinogen oxidase; 34) enhancing the expression of hemG, the gene encoding protoporphyrinogen oxidase; 36) introducing the gene encoding ferrochelatase hemH, the gene encoding heme oxygenase ho1, and the gene encoding glucose dehydrogenase gdhA; 38) The gene encoding phycocyanin ferredoxin oxidoreductase, pcyA, was introduced.

9. The recombinant Escherichia coli according to claim 8, characterized in that: The deposit number of the recombinant Escherichia coli is CGMCC No.32253.

10. A or B or C as follows: A is any of the following methods: A1) A method for constructing a recombinant Escherichia coli with improved 5-ALA production, comprising the following steps: carrying out the transformation according to 1) to 26) in claim 2 or 1) to 23) and 26) in claim 1; A2) A method for constructing a recombinant Escherichia coli with improved heme production, comprising the following steps: performing the transformation according to the transformations 1)-23), 26), and 27)-35) in claim 2; A3) A method for constructing a recombinant Escherichia coli with improved biliverdin production, comprising the following steps: carrying out the transformation according to 1)-23), 26), 27)-34), 36) in claim 4; A4) A method for constructing a recombinant Escherichia coli with improved bilirubin production, comprising the following steps: carrying out the transformation according to 1)-23), 26), 27)-34), 36) and 37) in claim 5; A5) A method for constructing a recombinant Escherichia coli with improved phycocyanin production, comprising the following steps: carrying out the transformation according to 1)-23), 26), 27)-34), 36) and 38) in claim 7; A6) A method for producing or increasing 5-ALA production, comprising the following steps: Cultivating the recombinant Escherichia coli according to claim 1 or 2 with glucose as a carbon source, collecting the bacterial cells; and then culturing the bacterial cells in a transformation medium of D-glucose to obtain 5-ALA; A7) A method for producing or increasing heme production, comprising the following steps: Cultivating the recombinant Escherichia coli according to claim 3 or 4 with glucose as a carbon source, collecting the bacterial cells; then culturing the bacterial cells in a transformation medium of D-glucose to obtain heme; A8) A method for producing or increasing the yield of biliverdin, comprising the following steps: Cultivating the recombinant Escherichia coli according to claim 5 with glycerol as a carbon source, collecting the bacterial cells; and then culturing the bacterial cells in a transformation medium containing D-glucose to obtain biliverdin; A9) A method for producing or increasing bilirubin production, comprising the steps of: Cultivating the recombinant Escherichia coli according to claim 6 or 7 with glycerol as a carbon source, collecting the bacterial cells; and then culturing the bacterial cells in a transformation medium containing D-glucose to obtain bilirubin; A10) A method for producing or increasing the yield of phycocyanin, comprising the following steps: Cultivating the recombinant Escherichia coli according to claim 8 or 9 with glycerol as a carbon source, collecting the bacterial cells; and then culturing the bacterial cells in a transformation medium containing D-glucose to obtain phycocyanin; B is any of the following applications: B1) Use of the recombinant Escherichia coli according to any one of claims 1 to 9 in the preparation, production or improvement of 5-ALA, heme, biliverdin, bilirubin or phycocyanin products; B2) Use of the recombinant Escherichia coli according to claim 1 or 2 in producing or increasing the yield of 5-ALA; B3) Use of the recombinant Escherichia coli according to claim 3 or 4 in producing or increasing heme production; B4) Use of the recombinant Escherichia coli according to claim 5 in producing or increasing the yield of biliverdin; B5) Use of the recombinant Escherichia coli according to claim 6 or 7 in producing or increasing the production of bilirubin; B6) Use of the recombinant Escherichia coli according to claim 8 or 9 in producing or increasing the yield of phycocyanin; C is any of the following products: C1) A product for producing or increasing the yield of 5-ALA, wherein the active ingredient is the recombinant Escherichia coli according to claim 1 or 2; C2) A product for producing or increasing the production of heme, wherein the active ingredient is the recombinant Escherichia coli according to claim 3 or 4; C3) A product for producing or increasing the production of biliverdin, wherein the active ingredient is the recombinant Escherichia coli according to claim 5; C4) A product for producing or increasing the production of bilirubin, wherein the active ingredient is the recombinant Escherichia coli according to claim 6 or 7; C5) A product for producing or increasing the yield of phycocyanin, wherein the active ingredient is the recombinant Escherichia coli according to claim 8 or 9.

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