CN115747125B - Engineering strain for high production of 5-aminolevulinic acid and construction method of 5-aminolevulinic acid high production strain - Google Patents

Abstract

The invention belongs to the technical field of microorganisms and biology, and particularly relates to a genetically engineered bacterium for producing 5-aminolevulinic acid in high yield and a method for producing 5-aminolevulinic acid by using the genetically engineered bacterium. The invention obtains the recombinant genetically engineered bacteria of high-yield 5-ALA through a series of metabolic engineering transformation. In a most preferred example, the yield of the 5-ALA of the obtained recombinant strain reaches 30.65 g/L, the production strength and the conversion rate are respectively 1.02 g/L and 0.53 mol/mol, the production cost of the 5-ALA can be greatly reduced, the requirement of industrial application can be met, and the recombinant strain has good application potential.

Description

Engineering strain with high production of 5-aminolevulinic acid and construction method of 5-aminolevulinic acid high production strain

Technical Field

The invention belongs to the field of microorganisms and biotechnology, and particularly relates to an Escherichia coli capable of producing high levels of 5-aminolevulinic acid, and a method for producing 5-aminolevulinic acid by using the strain.

Background technique

5-aminolevulinic acid (5-ALA) is a precursor of tetrapyrrole compounds such as heme, chlorophyll, and vitamin B12 in organisms. Tetrapyrrole compounds are important components of cytochromes, hemoglobin, chloroplast proteins, etc., and play an important role in life activities. Due to its characteristics of being degradable, non-toxic and residue-free, 5-ALA has broad application prospects in the fields of medicine, agriculture, animal husbandry, and daily chemicals, and is an important high-value-added bio-based chemical. As a new generation of photodynamic drugs, 5-ALA can be used for cancer treatment, tumor diagnosis, and treatment of skin diseases; as a plant growth regulator, 5-ALA can greatly promote the growth of flowers, crops, and vegetables, and improve the quality of crops, fruits, and vegetables; as an animal feed additive, 5-ALA can enhance the metabolism and immunity of animals. In recent years, 5-ALA has also been used as a main additive in the development of cosmetics and health foods, and related products have been put on the market.

However, currently 5-ALA is mainly prepared through chemical synthesis methods, which have disadvantages such as many reaction steps, low conversion rate, high production cost, high energy and material consumption, use of toxic raw materials in the preparation process, serious environmental pollution and high price.

With the development of society and science and technology, the production of 5-ALA by efficient and clean biological fermentation instead of chemical synthesis has become the focus of research. At present, there are two main methods for producing 5-ALA using microorganisms: one is to carry out mutation breeding of photosynthetic bacteria, select mutants with high 5-ALA production and culture them on specific culture media to accumulate high concentrations of 5-ALA, but this method has a long fermentation cycle, complex condition control, and the addition of substrates and inhibitors also increases production costs. The second is to use metabolic engineering technology to transform the metabolic pathways of microorganisms and to increase the excessive accumulation of 5-ALA by strengthening the synthesis of 5-ALA. The strains of microorganisms that synthesize 5-ALA are mainly Escherichia coli and Corynebacterium glutamicum. However, the current production capacity of 5-ALA by microorganisms is relatively low. For example, the highest yield reported for Escherichia coli is 15.6 g/L (Yu, T.-H., Tan, S.-I., Yi, Y.-C., Xue,C., Ting, W.-W., Chang, J.-J., Ng, IS, 2022. New insight into the codonusage and medium optimization toward stable and high-level 5-aminolevulinic acid production in Escherichia coli . Biochem. Eng. J. 177, 108259.); the highest yield for Corynebacterium glutamicum is 25.05 g/L (production intensity 0.52 g/L/h, conversion rate 16.79%) (Wang Lijun et al. Metabolic modification of recombinant Corynebacterium glutamicum C4 pathway for efficient synthesis of 5-aminolevulinic acid. Journal of Biotechnology, 2021, 37(12)). In addition, the fermentation cycle is long and the sugar-acid conversion rate is low, which makes it difficult to meet the requirements of industrial production.

In summary, there is an urgent need in this field to construct more efficient engineered strains in order to increase the yield of 5-ALA and further reduce production costs.

Summary of the invention

In response to the above needs, the present invention obtains recombinant Escherichia coli with high 5-ALA production through a series of metabolic engineering modifications.

The purpose of the present invention is to provide a method for constructing a 5-ALA high-yielding strain, a 5-ALA high-yielding strain constructed by the method, and a method for producing 5-ALA by using the strain.

In the first aspect, a method for constructing a 5-ALA high-yield strain is provided, characterized in that the activity of 5-aminolevulinic acid synthase in the strain is enhanced, the activity of phosphoenolpyruvate carboxylase is enhanced, the activity of succinyl-CoA synthetase is weakened, the activity of 5-aminolevulinic acid dehydratase is weakened, the activity of isocitrate lyase is weakened, the activity of pantothenate kinase is enhanced, the activity of 5-ALA transporter is enhanced, and the activity of proteins related to the 5-ALA oxidative damage repair system is enhanced.

Preferably, the enhancing the activity of 5-aminolevulinic acid synthase, the enhancing the activity of phosphoenolpyruvate carboxylase, the enhancing the activity of pantothenate kinase, the enhancing the activity of 5-ALA transporter, and the enhancing the activity of proteins related to the 5-ALA oxidative damage repair system are by increasing the copy number of polynucleotides encoding the protein, modifying the regulatory sequence of the gene encoding the protein, replacing the regulatory sequence of the gene encoding the protein on the chromosome with a sequence with strong activity, replacing the gene encoding the protein with a mutant gene, and introducing modifications in the gene encoding the protein on the chromosome to enhance the activity of the protein.

More preferably, the weakening of the activity of succinyl-CoA synthetase, the weakening of the activity of 5-aminolevulinic acid dehydratase, and the weakening of the activity of isocitrate lyase is achieved by partially or completely knocking out the enzyme's coding gene, inactivating or partially inactivating the gene by mutation, changing the gene promoter or translation regulatory region to weaken its transcription or translation, changing the gene sequence to weaken its mRNA stability or destabilize the enzyme structure, weakening the gene by sRNA, and weakening its activity by exogenously adding an inhibitor of enzyme activity.

Further preferably, the 5-ALA transporter is a cysteine/O-acetylserine transporter, and the 5-ALA oxidative damage repair system-related protein is glutaredoxin I. The amino acid sequence of the 5-aminolevulinic acid synthetase is as shown in SEQ ID No: 49, or the amino acid sequence of the 5-aminolevulinic acid synthetase is as shown in SEQ ID No: 49, wherein position 75 of the sequence is mutated from cysteine to alanine, and position 365 of the arginine is mutated to lysine.

More preferably, the strain is Escherichia coli or Corynebacterium glutamicum.

In the second aspect, the present invention provides a recombinant engineered strain with high 5-ALA production, characterized in that the strain has enhanced 5-aminolevulinic acid synthase activity, enhanced phosphoenolpyruvate carboxylase activity, weakened succinyl-CoA synthetase activity, weakened 5-aminolevulinic acid dehydratase activity, weakened isocitrate lyase activity, enhanced pantothenate kinase activity, enhanced 5-ALA transporter activity, and enhanced activity of proteins related to the 5-ALA oxidative damage repair system.

Preferably, the enhancing the activity of 5-aminolevulinic acid synthase, the enhancing the activity of phosphoenolpyruvate carboxylase, the enhancing the activity of pantothenate kinase, the enhancing the activity of 5-ALA transporter, and the enhancing the activity of proteins related to the 5-ALA oxidative damage repair system are by increasing the copy number of polynucleotides encoding the protein, modifying the regulatory sequence of the gene encoding the protein, replacing the regulatory sequence of the gene encoding the protein on the chromosome with a sequence with strong activity, replacing the gene encoding the protein with a mutant gene, and introducing modifications in the gene encoding the protein on the chromosome to enhance the activity of the protein.

More preferably, the weakening of the activity of succinyl-CoA synthetase, the weakening of the activity of 5-aminolevulinic acid dehydratase, and the weakening of the activity of isocitrate lyase is achieved by partially or completely knocking out the enzyme's coding gene, inactivating or partially inactivating the gene by mutation, changing the gene promoter or translation regulatory region to weaken its transcription or translation, changing the gene sequence to weaken its mRNA stability or destabilize the enzyme structure, weakening the gene by sRNA, and weakening its activity by exogenously adding an inhibitor of enzyme activity.

Further preferably, the 5-ALA transporter is a cysteine/O-acetylserine transporter, and the 5-ALA oxidative damage repair system-related protein is glutaredoxin I. The amino acid sequence of the 5-aminolevulinic acid synthetase is as shown in SEQ ID No: 49, or the amino acid sequence of the 5-aminolevulinic acid synthetase is as shown in SEQ ID No: 49, wherein position 75 of the sequence is mutated from cysteine to alanine, and position 365 of the arginine is mutated to lysine.

More preferably, the strain is Escherichia coli or Corynebacterium glutamicum.

In a third aspect, the present invention provides a method for producing 5-ALA, characterized in that the 5-ALA recombinant engineered strain is fermented and cultured to produce 5-ALA, and optionally, further comprises the step of separating 5-ALA from the culture medium.

Optionally, the seed medium for fermentation culture is Na ₂ HPO ₄ ·12H ₂ O 12.8 g/L, KH ₂ PO ₄ 3.0 g/L, NaCl 0.5 g/L, NH ₄ Cl 1.0 g/L, MgSO ₄ 2 mM, CaCl ₂ 0.1 mM, glucose 10 g/L, yeast powder 2 g/L, ampicillin 100 μg/mL and chloramphenicol 20 μg/mL.

More optionally, the fermentation medium of the fermentation culture is _{KH2PO4 5} g/L, _NH4Cl 8 g/L, yeast powder 5 g/L, _MgSO4 0.5 g/L, 0.01 g/L calcium pantothenate, defoamer 0.1 g/L and glucose 20 g/L.

The present invention obtains a recombinant Escherichia coli with high 5-ALA production through a series of metabolic engineering transformations. Among them, in an experimental verification in a most preferred example, the 5-ALA production of the obtained recombinant strain reached 30.65 g/L, and the production intensity and conversion rate were 1.02 g/L and 0.53 mol/mol, respectively, which are the highest levels reported so far, can greatly reduce the production cost of 5-ALA, can meet the needs of industrial applications, and has good application potential.

Instruction Manual

Figure 1 Testing the production capacity of the high-yielding strain ALA8 in a 5L fermenter.

Detailed ways

definition

The terms "comprising", "having", "including" or "containing" herein are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term "about" means that a value includes the standard deviation of error for the device or method used to determine the value.

The term "or" in the present invention is defined as only alternatives and "and/or", but unless it is clearly stated that there are only alternatives or the alternatives are mutually exclusive, the term "or" in the claims means "and/or".

When used in the claims or specification, a selected/optional/preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

The term "polynucleotide" of the present invention refers to a polymer composed of nucleotides. A polynucleotide may be in the form of a single fragment or may be a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence isolated at least once in quantity or concentration, and can be identified, manipulated, and recovered by standard molecular biology methods (e.g., using cloning vectors) and its component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), in which "U" replaces "T". In other words, a "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (single fragments or entire fragments), or may be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. "Recombinant polynucleotide" is a type of "polynucleotide".

The terms "sequence identity" and "percentage of identity" of the present invention refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the percentage of identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

In some embodiments, two or more sequences or subsequences have a "sequence identity" or "percent identity" of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotides when compared and aligned with maximum correspondence using a sequence comparison algorithm or as measured by visual inspection. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the compared biopolymers (e.g., polynucleotides).

The terms "polypeptide", "peptide" and "protein" of the present invention are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

The term "endogenous" of the present invention refers to a polynucleotide, polypeptide or other compound that is naturally expressed or produced in an organism or cell. That is, an endogenous polynucleotide, polypeptide or other compound is not exogenous. For example, an "endogenous" polynucleotide or polypeptide is present in a cell when the cell is initially isolated from nature.

The term "enhancing the activity of a protein compared to endogenous" in the present invention refers to enhancing the intracellular activity of a protein in a microorganism by modifying the protein to increase the intracellular activity compared to the activity of the protein in its natural state. The term "exogenous" as used herein refers to the inclusion of substances that did not originally exist in a system. For example, including but not limited to introducing a gene encoding an enzyme that did not originally exist in a strain into the strain by transformation, thereby expressing the enzyme in the strain, the enzyme is "exogenous" to the strain.

The term "enhancing the activity of a protein" of the present invention not only includes effects higher than the original function due to the increase in the activity of the protein itself, but can also be achieved by at least one method selected from the following: increasing the copy number of a polynucleotide encoding a protein, modifying the regulatory sequence of a gene encoding a protein, replacing the regulatory sequence of a gene encoding a protein on a chromosome with a sequence having strong activity, replacing a gene encoding a protein with a mutant gene to increase the activity of the protein, introducing modifications into a gene encoding a protein on a chromosome to enhance the activity of the protein, and can also include, without limitation, any existing method, as long as it can enhance the activity of the protein or enhance the activity of the introduced protein compared to the endogenous activity.

The term "introducing protein activity" of the present invention has the meaning conventionally understood by those skilled in the art, and can be implemented by methods known in the art, including but not limited to: inserting a polynucleotide comprising a polynucleotide sequence encoding a protein into a chromosome, and/or cloning the polynucleotide into a vector and introducing it into a microorganism, and/or directly increasing the copy number of the polynucleotide on the chromosome, and/or transforming a polynucleotide promoter having a polynucleotide encoding a protein to enhance the transcription initiation rate, and/or modifying the transcription of a polynucleotide encoding a protein to enhance its activity, and/or modifying the translation regulatory sequence of a messenger RNA carrying the polynucleotide encoding the protein to enhance the translation intensity, and/or modifying the polynucleotide encoding the protein itself to enhance mRNA stability, protein stability, relieve protein feedback inhibition, etc., and can also include, without limitation, any known method for introducing protein activity.

As described above, the regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for transcriptional regulation, a sequence encoding a suitable mRNA ribosome binding domain, and a sequence regulating transcription and translation termination. Modifications to the regulatory sequence include, but are not limited to, deletions, insertions, conservative mutations or non-conservative mutations, or combinations thereof, in the polynucleotide sequence, and may also be introduced by replacing the original polynucleotide sequence with a polynucleotide sequence having enhanced activity.

Similarly, the terms "weaken" and "weakening" of the present invention refer to reducing, weakening, reducing or completely eliminating a certain protein, such as the activity of an enzyme. In a specific embodiment, the activity of an enzyme can be weakened by partially or completely knocking out the coding gene of the enzyme, inactivating or partially inactivating a gene mutation, changing a gene promoter or a translation control region to weaken its transcription or translation, changing the gene sequence to weaken its mRNA stability or the enzyme structure is unstable, regulating and controlling the gene by sRNA, and other methods or combinations thereof, and can also be achieved by exogenously adding an inhibitor of enzyme activity, including but not limited to the above method. Based on the teachings of the present invention, those skilled in the art can also use similar expressions such as "partial deletion", "deletion", "closing", "inactivation" to represent "weakening".

The term "5-aminolevulinic acid synthetic pathway" of the present invention refers to a specific pathway for producing 5-aminolevulinic acid by a carbon 4 (C4) pathway in a microorganism, including various enzymes, such as 5-aminolevulinic acid synthetase and the like. Similarly, the term "5-aminolevulinic acid synthetic pathway enhancement" used herein refers to an activity enhancement of an enzyme related to a carbon 4 (C4) pathway, such as 5-aminolevulinic acid synthetase. In a preferred embodiment, the enzyme includes but is not limited to a 5-aminolevulinic acid synthetase derived from Rhodopseudomonas palustris.

The term "wild-type" of the present invention refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory is naturally occurring. As used herein, "naturally occurring" and "wild-type" are synonyms.

The term "mutant" of the present invention refers to a polynucleotide or polypeptide comprising a change (i.e., substitution, insertion and/or deletion) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

The term "mutated amino acid" of the present invention includes "substitution, duplication, deletion or addition of one or more amino acids". In the present invention, the term "mutation" refers to a change in the amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

In one embodiment, the "mutation" of the present invention can be selected from "conservative mutations". In the present invention, the term "conservative mutation" refers to a mutation that can maintain the function of a protein normally. A representative example of a conservative mutation is a conservative substitution.

The term "conservative substitution" of the present invention relates to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, and include having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). As used in the present invention, "conservative substitution" is usually exchanged for an amino acid at one or more sites of a protein. This substitution can be conservative. As a substitution considered as a conservative substitution, in addition, conservative mutations also include naturally occurring mutations resulting from individual differences, strains, and differences in species from which genes are derived.

The term "corresponding to" in the present invention has the meaning generally understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, a position in one sequence corresponds to a specified position in another sequence. Thus, for example, with respect to "the amino acid residue corresponding to position 150 of the amino acid sequence shown in SEQ ID NO. 1", if a 6×His tag is added to one end of the amino acid sequence shown in SEQ ID NO. 1, then the position corresponding to position 150 of the amino acid sequence shown in SEQ ID NO. 1 in the resulting mutant may be position 156.

The term "expression vector" of the present invention refers to a DNA construct containing a DNA sequence operably linked to an appropriate control sequence so as to express a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired exogenous polypeptide. The recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and iii) a transcription subunit that is suitable for transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. For example, as a plasmid vector, pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. can be used, specifically, pTrc99A, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pXMJ19 vectors, etc. can be used, but not limited to this, as long as it can be replicated and expressed in Corynebacterium glutamicum. As a phage vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used.

The term "recombinant engineered strain" in the present invention covers strains different from parent cells produced by introducing exogenous polynucleotides, nucleic acid constructs or recombinant expression vectors through various genetic modification methods, knocking out proteins in wild-type strains, etc. The recombinant engineered strain is specifically achieved through transformation.

The term "transformation" in the present invention has the meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The transformation method includes any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.

The culture of the recombinant engineered strain of the present invention can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and batch fed culture, and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

The 5-aminolevulinic acid production strain constructed by the present invention is a 5-aminolevulinic acid high-yield strain. Those skilled in the art know that many strains can be used to produce 5-aminolevulinic acid. Although these strains are different, their synthesis systems and pathways for synthesizing 5-aminolevulinic acid are similar. Based on the teachings of the present invention and the prior art, those skilled in the art can implement the present invention using various suitable strains, including but not limited to Escherichia coli , Corynebacterium glutamicum , Rhodobactersphaeroides , Rhodopseudomonas palustris , etc.; preferably Escherichia coli or Corynebacterium glutamicum, most preferably Escherichia coli. Based on the method for constructing 5-aminolevulinic acid provided by the present invention, those skilled in the art can use any Escherichia coli or Corynebacterium glutamicum to carry out the above transformation to obtain a 5-aminolevulinic acid high-yield strain, which is obvious.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.

The primers used in the embodiments of the present invention are as follows:

Primer name Sequence (5'-3') SEQ ID No pZCA9-F GCCTGGGGTGCCTAATGAGT 1 pZCA9-R TCACACAGGAAACAGCTATGAC 2 Weakening the sRNA of the sucC gene encoding succinyl-CoA synthetase TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACTGCCTGATATTCATGTAAGTTCATTTTCTGTTGGGCCATTGCATTGCCACTGATTTTCCAACATATAAAAAGACAAGCCCGAACAGTCGTCCGGGCTTTTTTTCTCGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC 3 TsR TCCCTATCAGTGATAGAGATTGAC 4 TsR GAGAGCGTTCACCGACAAACA 5 tetR-F TTGTCGGTGAACGCTCTCGGGTCGACTCTAGAGGATCT 6 tetR-R CCTGATTCTGTGGATAACCGGCTTTTAAGACCCACTTTCA 7 p-F CGGTTATCCACAGAATCAGG 8 p-R TCTCTATCACTGATAGGGACGCCTTTGAGTGAGCTGATAC 9 sRhemB-F TAAGTCTGTCATTTTCTGTTGGGCCATTGCAT 10 sRhemB-R ATCCAACGCCCTGTGCTCAGTATCTCTATCACTGA 11 sRaceA-F ACGGGTTTTCATTTTCTGTTGGGCCATTGCAT 12 sRaceA-R ACACAACAAATTGTGCTCAGTATCTCTATCACTGA 13 TsRBC-F TTTGTCGGTGAACGCTCTCTAATGAATCGGCCAACGCG 14 TsRBC-R TCTCTATCACTGATAGGGATAATGCAGCTGGCACGACA 15 TsRAC-F TTTGTCGGTGAACGCTCTCAGCGGTATCAGCTCACTCAA 16 TsRAC-R TCTCTATCACTGATAGGGACCAATACGCAAACCGCCTCT 17 coaA- F TCCGCTTACAGACAAGCTGTTTATTTGCGTAGTCTGACCTCTTC 18 coaA- R TGACGGTGAAAACCTCTGACTTGACGGCTAGCTCAGTCCT 19 pTrc99a-Rev-2F GTCAGAGGTTTTCACCGTCAT 20 pTrc99a-Rev-2R ACAGCTTGTCTGTAAGCGGA twenty one ppc -F CCGC AAGCTT TATCCGACCTACACCTTTGGT twenty two ppc -R CCGC AAGCTT GGACTTCTGTGGAATGCATAGT twenty three C75A-F GACAGCGCCGGCGCCGGCCGGCCGGCGGCAC twenty four C75A-R GCCGGCGCCGGCGCTGTCCAGCGCCTCGTG 25 R365K-F GTGCCGAAAGGCACCGAGCGCCTTCGGATCAC 26 R365K-R CGGTGCCTTTCGGCACGGTCGGATAGTTGATC 27 eamA- F CAGAACGCAGAAGCGGTCTGAAAGAGCACAACCAGCGTCA 28 eamA- R GCCGCCAGGCAAATTCTGTTTTAACTTCCCACCTTTACCGCT 29 hemA -F ACCAATGCTTCTGGCGTCA 30 ppc -2R CAGACCGCTTCTGCGTTCT 31 amp -F AACAGAATTTGCCTGGCGG 32 lacI -R TGACGCCAGAAGCATTGGT 33 grxA -F CGGTAAAGGTGGGAAGTTAATCATTCTGGCAAGAGCTGGC 34 grxA -R GCCGCCAGGCAAATTCTGTTACCTGAAGAATAACCACCACCG 35 eamA -2R TTAACTTCCCACCTTTACCGCT 36 coaA -500-F AGTCTGTGACGGCAGATTTA 37 coaA -500-R TGCAGATAAAGTTCGTGTTAA 38 coaA -Rev-F TTTGCAGGGGAGCGAATACT 39 coaA -Rev-R GGTTTAGCAGGAGCGCATTG 40 cm-F GGTGAAAACCTCTGACACATGCAG 41 SacB-R CGCCAGTGTGCTGGAATTGC 42 pKD46- coaA -F CAAATAGGGGTTCCGCGCACTCAACGATAGCTTCCTGGCAGAGAT 43 pKD46- coaA -R GTGGCACTTTTCGGGGAAATTTATTTGCGTAGTCTGACCT 44 pKD46-Rev-F ATTTCCCCGAAAAGTGCCACCTG 45 pKD46-Rev-R GTGCGCGGAACCCCTATTTGTT 46 coaA -TF GAGAAGCTCTCTTTATTCGAGCCG 47 coaA -TR ATTTGTCGATGTTCTGGCGTCTGG 48

Example 1. Construction of vector for weakening the competitive pathway of 5-ALA synthesis based on sRNA

In order to weaken the competitive pathway for the supply of succinyl-CoA, the precursor of 5-ALA synthesis, a gene expression weakening tool based on sRNA was designed. First, the plasmids pSB4C5 (Shetty, RP, Endy, D., Knight, TF, Jr., 2008. Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2, 5.) and pWSK29 (Wang, RF, Kushner, SR, 1991. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli . Gene 100, 195-199.) were double-digested with Nde I and Eco RI, respectively, to obtain a fragment carrying the chloramphenicol resistance gene cat and the low-copy pSC101 replicon, and the two fragments were recombined to obtain the low-copy cloning vector pZCA9. The pZCA9 vector was reversely amplified using pZCA9-F/R primers, the lac promoter was removed, and the obtained fragment was self-circularized to form the pZCA9P vector for loading the sRNA fragment.

According to the sRNA tool reported in the literature (Yoo, SM, Na, D., Lee, SY, 2013. Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli . Nat. Protoc. 8, 1694-1707.), the sRNA (SEQ ID No: 3) of the weakened succinyl-CoA synthetase encoding gene sucC (Genebank ID: 945312) was designed and synthesized. The sequence includes the promoter of the TetR regulatory sequence, the N-terminal complementary pairing region of the sucC gene, the MicF Scaffold region and the T1/TE terminator. The above gene fragment was fully synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the vector pUC57 to obtain the plasmid pUC57-sRNA. The vector pUC57-sRNA carrying the above sRNA sequence was used as a template, and the universal primers TsR-F/R were used to amplify the sRNA fragment; at the same time, the pACYC184 (Rose, RE, 1988. The nucleotide sequence of pACYC184. Nucleic AcidsRes. 16, 355.) vector was used as a template, and the tetR gene fragment was amplified using the tetR-F/R primers. The linearized vector fragment was obtained by reverse amplification using pF/R primers using pZCA9P as a template. The above three fragments were recovered and recombined to obtain the attenuated vector pZSA14 of the sucC gene.

Using pZSA14 as a template, sRhemB-F/R and sRaceA-F/R primers were used for reverse amplification and self-cyclization to obtain the weakened vectors pZSA13 and pZSA41 of the 5-aminolevulinic acid dehydratase encoding gene hemB (GenebankID: 945017) and the isocitrate lyase encoding gene aceA (GenebankID: 948517), respectively. Using pZSA13 as a template, the universal primers TsR-F/R were used to amplify the weakened sRNA fragment of the hemB gene, and using pZSA14 as a template, the TsRBC-F/R primers were used for reverse amplification to obtain the linearized plasmid fragment. The two fragments were recombined to obtain the vector pZCA134 with both sucC and hemB weakened. Using pZSA41 as template, universal primers TsR-F/R were used to amplify the weakened sRNA fragment of aceA gene. Using pZSA134 as template, TsRAC-F/R primers were used to reversely amplify the linearized plasmid fragment. The two fragments were recombined to obtain the vector pZCA136 with weakened sucC , hemB and aceA at the same time.

Example 2. Construction of pantothenate kinase expression vector

Primers coaA -F and coaA -R were designed according to the genome sequence of Escherichia coli MG1655 published by NCBI. The coaA (Genebank ID: 948479) fragment encoding the pantothenate kinase gene with the promoter P _J23100 (5'-TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC-3') was amplified by PCR using the Escherichia coli MG1655 genome as a template. Using plasmid pTrc99A- hemA (abbreviated as pZGA24) (Zhang, L., Chen, J., Chen, N., Sun, J., Zheng, P. and Ma, Y.2013. Cloning of two 5-aminolevulinic acid synthase isozymes HemA and HemOfrom Rhodopseudomonas palustris with favorable characteristics for 5-aminolevulinic acid production. Biotechnology letters, 35, 763-768.) as a template, primers pTrc99A-Rev-2F/pTrc99A-Rev-2R were used to reversely amplify the plasmid to obtain the corresponding linearized vector fragment. After the above two fragments were purified and recovered, the coaA overexpression vector pTrc99A- hemA-coaA was obtained by recombinase ligation and named pZPW70.

Example 3. Construction of phosphoenolpyruvate carboxylase PPC expression vector

Primers ppc -F/ ppc -R were designed based on the genome sequence of Escherichia coli MG1655 published by NCBI. The ppc (Genebank ID: 948457) fragment encoding the phosphoenolpyruvate carboxylase gene was amplified by PCR using the Escherichia coli MG1655 genome as a template. The ppc gene fragment and vector pZPW70 were digested with restriction endonuclease HindⅢ , and the digested product of vector pZPW70 was dephosphorylated using dephosphorylase. After the two fragments were purified and recovered, the vector fragment and the target gene fragment were connected together using T4 DNAligase. After transformation, restriction digestion and sequencing verification, the correct recombinant vector pTrc99A- hemA-ppc-coaA was obtained and named pZPW71.

Example 4. Construction of 5-ALA synthase mutant expression vector

According to the sequence information of plasmid pZPW71, the site-directed mutagenesis primers C75A-F/C75A-R of the gene hemA encoding 5-aminolevulinic acid synthase (SEQ ID No: 49) were designed, and the correct recombinant vector pTrc99A- hemA ^C75A - ppc-coaA was obtained by PCR reverse amplification, fragment purification and ligation transformation, restriction digestion and sequencing verification. Using plasmid pTrc99A- hemA ^C75A - ppc- coaA as a template, site-directed mutagenesis primers R365K-F/R365K-R were designed, and the correct recombinant vector pTrc99A- hemA ^C75A/R365K - ppc-coaA was verified by PCR reverse amplification, fragment purification and ligation transformation, restriction digestion and sequencing and named pZPW72.

Example 5. Construction of 5-ALA transporter expression vector

Primers eamA -F/ eamA -R were designed based on the genome sequence of Escherichia coli MG1655 published by NCBI. The 5-ALA transporter (cysteine/O-acetylserine transporter) encoding gene eamA (GenebankID: 946081) fragments with the promoter on the genome were amplified by PCR using the Escherichia coli MG1655 genome as a template. Plasmid backbone fragment 1 ( hemA ^C75A/R365K + ppc gene fragment) and plasmid backbone 2 ( bla + coaA + lacI gene fragment) were amplified using plasmid pZPW72 as a template and primers hemA -F/ ppc -2R and amp -F/ lacI -R , respectively. After the above fragments were purified and recovered, they were ligated with recombinase, and the correct recombinant vector pTrc99A- hemA ^C75A/R365K - ppc-coaA - eamA was obtained by transformation, restriction digestion and sequencing verification, and named pZPW73.

Example 6. Construction of gene expression vector related to 5-ALA oxidative damage repair system

According to the genome sequence of Escherichia coli MG1655 published by NCBI, primers grxA- F/ grxA -R were designed. Using the genome of Escherichia coli MG1655 as a template, PCR amplification was performed to obtain the gene fragment of grxA (GenebankID: 945479), which encodes glutaredoxin I, with a promoter on its own genome. Plasmid pZPW72 was used as a template, and plasmid backbone fragment 1 ( hemA ^C75A/R365K + ppc gene fragment) and plasmid backbone 2 ( bla + coaA + lacI gene fragment) were amplified using primers hemA -F/ ppc -2R and amp -F/ lacI -R, respectively. According to the genome sequence of Escherichia coli MG1655 published by NCBI, primers eamA -F/ eamA -2R were designed. Using the genome of Escherichia coli MG1655 as a template, PCR amplification was performed to obtain the eamA gene fragment with a promoter on its own genome. After the above four fragments were purified and recovered, they were connected using recombinase, and the correct recombinant vector pTrc99A- hemA ^C75A/R365K - ppc-coaA - eamA - grxA was obtained through transformation, enzyme digestion and sequencing verification, and named pZPW76.

Example 7. Using sRNA to weaken the competition pathway for 5-ALA synthesis and enhance 5-ALA accumulation

The attenuated plasmid pZCA136 of the 5-ALA synthetic competitive pathway constructed in Example 1 and the empty vector control pZCA9 were transformed into the strain E. coli MG1655 (pZGA24) (see CN103981203A for this strain), and the correct engineered strains MG1655 (pZGA24 + pZCA9) and MG1655 (pZGA24 + pZCA136) were obtained by PCR verification, and the strains were named ALA1 and ALA2, respectively. The recombinant bacteria ALA1 and ALA2 single colonies were inoculated into 24-well plates containing 1 mL LB liquid culture medium (100 μg/mL ampicillin and 20 μg/mL chloramphenicol were added to the culture medium) and cultured at 37°C and 800 rpm for 8 hours. According to the initial OD _600nm of 0.05, the culture was transferred to a 24-well plate containing 1 mL of 5-ALA fermentation medium and cultured at 37°C and 800 rpm. After culturing for 2.5 h, IPTG with a final concentration of 25 μM, 4 g/L glycine and 100 ng/mL dehydrotetracycline were added, and the temperature was lowered to 35°C. After a total of 24 h of culture, the fermentation was terminated, the fermentation broth was collected, and the OD _600nm , 5-ALA concentration and residual sugar were detected. The 5-ALA fermentation medium is M9 medium added with a small amount of yeast powder, and its main components are: Na ₂ HPO ₄ ·12H ₂ O 12.8 g/L, KH ₂ PO ₄ 3.0 g/L, NaCl 0.5 g/L, NH ₄ Cl 1.0 g/L, MgSO ₄ 2 mM, CaCl ₂ 0.1 mM, glucose 15 g/L, yeast powder 2 g/L, ampicillin 100 μg/mL and chloramphenicol 20 μg/mL.

5-ALA detection method: 200 μL of diluted fermentation broth was added with 100 μL of pH 4.6 sodium acetate buffer, and then 5 μL of acetylacetone was added, and the mixture was incubated in a water bath at 100°C for 15 min. After cooling to room temperature, an equal volume of Ehrlish's reagent (42 mL of glacial acetic acid, 8 mL of 70% perchloric acid, 1 g of dimethylaminobenzaldehyde) was added and mixed, and the absorbance at OD _{553 nm} was measured after color development for 10 min. The yield of each recombinant strain is shown in Table 1. The 5-ALA yield of the ALA2 strain was increased to 1.56 g/L, which was 26.8% higher than that of the control strain ALA1.

Table 1 Effects of weakening the competitive pathway of 5-ALA synthesis by sRNA on 5-ALA accumulation

Strains 5-ALA (g/L) OD _600nm ALA1 1.23 ± 0.03 6.34 ± 0.27 ALA2 1.56 ±0.038 6.62 ± 0.21

Example 8. Enhancing the expression of pantothenate kinase to enhance 5-ALA accumulation

The co-overexpression vectors pZPW70 and pZCA136 of coaA and hemA constructed in Example 2 were co-transformed into the strain E. coli MG1655. After PCR verification, the correct engineered strain MG1655 (pZPW70 + pZCA136) was obtained, and the strain was named ALA3. ALA3 and the control strain ALA2 were cultured according to the 5-ALA well plate fermentation process in Example 7 (the culture medium of this example is the culture medium of Example 7 with 0.2 g/L of calcium pantothenate added, and other culture conditions are the same). The yield of the recombinant bacteria is shown in Table 2, and it can be seen that the 5-ALA yield of ALA3 is 2.44 g/L, which is 33.3% higher than that of the control strain ALA2.

Table 2 Effect of enhanced intracellular CoA supply on 5-ALA accumulation

Strains 5-ALA (g/L) OD _600nm ALA2 1.83± 0.06 5.29± 0.02 ALA3 2.44± 0.12 5.56± 0.12

Example 9. Strengthening the four-carbon replenishment pathway to enhance the extracellular accumulation of 5-ALA

The ppc , coaA and hemA co-expression vector pZPW71 constructed in Example 3 was co-transformed into the strain E. coli MG1655 together with pZCA136. After PCR verification, the correct engineered strain MG1655 (pZPW71 + pZCA136) was obtained, and the strain was named ALA4. ALA4 and the control strain ALA3 were cultured according to the well plate culture process in Example 8 (the culture conditions of this example are based on the culture conditions of Example 8, and 10 g/L glucose and 2 g/L glycine are added after 12 hours of culture, and other conditions are the same). The yield of the recombinant bacteria is shown in Table 3. It can be seen that the 5-ALA yield and OD _600nm of ALA4 reached 4.05 g/L and 8.19, respectively, which are 66% and 47.3% higher than the control strain ALA3. The results show that strengthening the four-carbon replenishment pathway not only helps to increase 5-ALA accumulation, but also helps to strengthen the growth of the strain.

Table 3 Effect of overexpression of PPC on 5-ALA synthesis

Strains 5-ALA (g/L) OD _600nm ALA3 2.44 ± 0.12 5.56 ± 0.12 ALA4 4.05 ± 0.17 8.19± 0.07

Example 10. Protein engineering of 5-ALA synthase to improve 5-ALA accumulation

The hemA ^C75A/R365K constructed in Example 4 and the co-overexpression vectors pZPW72 and pZCA136 for ppc and coaA were co-transformed into the strain E. coli MG1655. After PCR verification, the correct engineered strain MG1655 (pZPW72 + pZCA136) was obtained, and the strain was named ALA5. ALA5 and the control strain ALA4 were cultured according to the well plate culture process in Example 9.

The yield of the recombinant strain is shown in Table 4. It can be seen that the 5-ALA yield of ALA5 reached 4.91 g/L, which was 21% higher than that of the control strain ALA4. This result shows that overexpression of 5-ALA synthase mutants with enhanced activity and relief of heme inhibition can help further improve the accumulation of 5-ALA.

Table 4 Effects of protein engineering of ALAS on 5-ALA synthesis

Strains 5-ALA (g/L) OD _600nm ALA4 4.05 ± 0.17 8.19 ± 0.07 ALA5 4.91 ± 0.19 8.78 ± 0.01

Example 11. Strengthening the transport pathway of 5-ALA to increase 5-ALA accumulation

The 5-ALA transporter encoding gene eamA constructed in Example 5 and the co-overexpression vector pZPW73 of ppc , coaA and hemA ^C75A/R365K were co-transformed into the strain E. coli MG1655 together with pZCA136. After PCR verification, the correct engineering strain MG1655 (pZPW73 + pZCA136) was obtained, and the strains were named ALA6. ALA6 and the control strain ALA5 were cultured according to the well plate culture process in Example 9. The yield of the recombinant bacteria is shown in Table 5, and it can be seen that the 5-ALA yield of ALA6 reached 5.69 g/L, which is 16% higher than that of the control strain ALA5. The results show that strengthening the transport pathway of 5-ALA helps to further improve the accumulation of 5-ALA.

Table 5 Effects of strengthening the transport pathway of 5-ALA on the accumulation of 5-ALA

Strains 5-ALA (g/L) OD _600nm ALA5 4.90 ± 0.02 10.39± 0.33 ALA6 5.69± 0.04 6.76± 0.04

Example 12. Effect of strengthening the oxidative damage repair system on 5-ALA accumulation

The co-overexpression vector pZPW76 of the oxidative damage repair system-related genes grxA , eamA , ppc , coaA and hemA ^C75A/R365K constructed in Example 6 was co-transformed into the strain E. coli MG1655 together with pZCA136. After PCR verification, the correct engineered strain MG1655 (pZPW76 + pZCA136) was obtained, and the strain was named ALA7. The ALA7 strain and the control strain ALA6 were cultured according to the well plate culture process in Example 8 (the culture conditions in this example are based on the culture conditions in Example 8, and 15 g/L glucose and 4 g/L glycine are added after 12 hours of culture, and other conditions are the same).

The yield of the recombinant strain is shown in Table 6. It can be seen that the 5-ALA yield of ALA7 reached 8.32 g/L, which was 21% higher than that of the control strain ALA6. These results indicate that strengthening the oxidative damage repair system of 5-ALA helps to increase 5-ALA accumulation.

Table 6 Effect of strengthening the oxidative damage repair system on 5-ALA accumulation

Strains 5-ALA (g/L) OD _600nm ALA6 6.87 ± 0.31 7.70± 0.10 ALA7 8.32± 0.04 8.37± 0.08

Example 13. Construction of ALA plasmid stable strain

First, the genome of E. coli MG1655 was used as a template, and the coaA gene and its upstream and downstream 500 bp fragments were amplified by PCR using primers coaA -500-F/ coaA -500-R. After the PCR amplification product was recovered, it was ligated with plasmid pEASY-Blunt (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the vector pEASY-Blunt-500- coaA -500, named pZPA17. Using plasmid pZPA17 as a template, the coaA gene was removed by PCR reverse amplification using primers coaA -Rev-F/ coaA -Rev-R to obtain a linearized vector fragment; the vector fragment PCR product was treated with restriction endonuclease Dpn I, and the fragment treated with Dpn I was further phosphorylated using T4 polynucleotide kinase, and then self-ligated using T4 DNA ligase to obtain the vector pZPW18. At the same time, plasmid pDS132 (Philippe, N., Alcaraz, JP, Coursange, E., Geiselmann, J., Schneider, D., 2004. Improvement of pCVD442, a suicide plasmid for geneallele exchange in bacteria. Plasmid 51, 246-255.) was used as a template, and primers cm-F/sacB-R were used to amplify cam and sacB gene fragments, which were ligated with the reverse amplified fragment of pZPA17 treated with phosphate to obtain vector pZPA19. Plasmid pZPA19 and pZPW18 were used as templates, and primers coaA -500-F/ coaA -500-R were used to amplify target fragment 1 (500 bp + cm-sacB + 500 bp) and target fragment 2 (500 bp fragments upstream and downstream of the coaA gene) of coaA gene knockout by PCR.

Using the genome of E. coli MG1655 as a template, primers pKD46 -coaA- F / pKD46 -coaA- R were used to amplify the coaA gene and its own promoter fragment by PCR, and the PCR product was further phosphorylated using T4 polynucleotide kinase. Using plasmid pKD46 as a template, primers pKD46-Rev-F/pKD46-Rev-R were used for reverse amplification by PCR to obtain a linearized vector fragment. Finally, the above two fragments were connected using T4 DNA ligase to obtain the vector pKD46- coaA , named pZPA43.

The chromosomal coaA gene was knocked out using the Red scarless recombination method reported in the literature (Datsenko, KA, Wanner, BL, 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645. and Philippe, N., Alcaraz, JP, Coursange, E., Geiselmann, J., Schneider, D., 2004. Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid 51, 246-255.). First, the auxiliary plasmid pZPA43 was transformed into the strain MG1655 by electric pulse to prepare the competent cells. The targeting fragment 1 with coaA gene knockout was transformed into the above-mentioned competent cells, and after culture, the primers coaA -TF/ coaA -TR were used for verification to obtain the first recombinant strain MG1655( △coaA::cam- sacB )/pZPA43; competent cells of strain MG1655( △coaA::cam-sacB )/pZPA43 were prepared, and the targeting fragment 2 was transformed into the competent cells. After culture and counter-screening with sucrose medium, the secondary recombinant strain MG1655( △coaA )/pZPA43 was obtained, which was named ZLEcA4. Competent cells of strain ZLEcA4 were prepared, plasmids pZCA136 and pZPA76 were co-transformed into strain ZLEcA4, and then cultured at 37°C with shaking for 18 h to remove the temperature-sensitive plasmid pZPA43, thereby obtaining strain MG1655 ( △coaA ) (pZCA136 + pZPA76), which was named ALA8.

Example 14. ALA plasmid stability strain 5 L fermentation tank fermentation

The production capacity of 5-ALA high-yielding strain ALA8 was tested in a 5L fermenter. The seed medium composition was: Na ₂ HPO ₄ ·12H ₂ O 12.8 g/L, KH ₂ PO ₄ 3.0 g/L, NaCl 0.5 g/L, NH ₄ Cl 1.0 g/L, MgSO ₄ 2 mM, CaCl ₂ 0.1 mM, glucose 10 g/L, yeast powder 2 g/L, ampicillin 100 μg/mL and chloramphenicol 20 μg/mL. The fermentation medium composition was: KH ₂ PO ₄ 5 g/L, NH ₄ Cl 8 g/L, yeast powder 5 g/L, MgSO ₄ 0.5 g/L, 0.01 g/L calcium pantothenate, defoamer 0.1 g/L and glucose 20 g/L.

The fermentation culture was carried out according to the process described in reference (Zhu, C., et al. Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli .Biotechnol. Bioeng. 116, 2018-2028.), during which glucose and glycine were added. After 30 h of fermentation culture, the yield of 5-ALA reached 30.65 g/L, and the production intensity and conversion rate were 1.02 g/L/h and 0.53%, respectively.

The highest levels reported in the literature are 15.6 g/L (Escherichia coli) (Zhu, C., et al. Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli . Biotechnol. Bioeng. 116, 2018-2028.) and 25.05 g/L (Corynebacterium glutamicum, production intensity 0.52 g/L/h, conversion rate 16.79%) (Wang Lijun et al. Metabolic modification of recombinant Corynebacterium glutamicum C4 pathway for efficient synthesis of 5-aminolevulinic acid. Journal of Biotechnology, 2021, 37(12)). The yield, conversion rate and production intensity of the Escherichia coli 5-ALA production bacteria constructed by the present invention are significantly higher than those reported in the literature, there is a synergistic effect of the modified genes, and it has unexpected technical effects, so it can significantly reduce the production cost of 5-ALA and meet the needs of industrial production.

Claims (9)

1. A method for constructing a high-yield engineering strain of 5-ALA, characterized in that the activity of 5-aminolevulinic acid synthase, phosphoenolpyruvate carboxylase, pantothenate kinase, cysteine/O-acetylserine transporter, glutaredoxin I, and the activity of succinyl-coa synthase, 5-aminolevulinic acid dehydratase, and isocitrate lyase are enhanced in the strain, and the activity of succinyl-coa synthase, 5-aminolevulinic acid dehydratase, and isocitrate lyase are attenuated or knocked out.

2. The method of claim 1, wherein the enhancement of activity of 5-aminolevulinic acid synthase, phosphoenolpyruvate carboxylase, pantothenate kinase, cysteine/O-acetylserine transporter, glutaredoxin I is achieved by increasing the copy number of a polynucleotide encoding a protein, modifying a regulatory sequence of a gene encoding a protein, replacing a regulatory sequence of a gene encoding a protein on a chromosome with a sequence having strong activity, replacing a gene encoding a protein with a mutant gene and/or introducing modifications in a gene encoding a protein on a chromosome to enhance the activity of a protein.

3. The method of claim 1, wherein the attenuation of the activity of succinyl-coa synthetase, 5-aminolevulinic acid dehydratase, isocitrate lyase is by partial or total knockout of the gene encoding the enzyme, inactivation of the gene mutation or partial inactivation, alteration of the gene promoter or translation regulatory region to attenuate transcription or translation, alteration of the gene sequence to attenuate mRNA stability or destabilize the enzyme structure, attenuation of the gene by sRNA and/or attenuation of the activity by exogenously added inhibitors of the enzyme.

4. The method of claim 1, wherein the 5-aminolevulinic acid synthase has an amino acid sequence as set forth in SEQ ID No:49 or the amino acid sequence of the 5-aminolevulinic acid synthase is as shown in SEQ ID No:49 from cysteine to alanine and from arginine to lysine at position 365.

5. The method of any one of claims 1 to 4, wherein the strain is escherichia coli (ESCHERICHIA COLI) or corynebacterium glutamicum (Corynebacterium glutamicum).

6. A recombinant engineering strain with high 5-ALA yield prepared by the method of any one of claims 1 to 5.

7. A method for producing 5-ALA, wherein the recombinant 5-ALA engineering strain according to claim 6 is cultured by fermentation to produce 5-ALA.

8. The method of claim 7, further comprising the step of isolating 5-ALA from the culture medium.

9. The method of claim 7 or 8, wherein the fermentation medium of the fermentation culture is KH ₂PO₄ 5 g/L,NH₄ Cl 8 g/L, yeast powder 5 g/L, mgSO ₄ 0.5.5 g/L,0.01 g/L calcium pantothenate, defoamer 0.1 g/L and glucose 20 g/L.