CN118064475B - A 5-aminolevulinic acid producing strain and its construction method and application - Google Patents

Abstract

The invention provides a 5-aminolevulinic acid producing strain and a construction method and application thereof. The strain upregulates aceF, pykF, icd, gdhA, gltx, hemA, heml and rhtA genes , downregulates hemB gene through BBa_J23113 promoter, heterologously expresses NCU05151 gene derived from Neurospora crassa , and carries the above-mentioned high-copy plasmid pETJAL, on which aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE genes of E.coli W3110 and alaS gene derived from Rhodobactersphaeroides are expressed; the strain realizes efficient production of 5-aminolevulinic acid and has excellent industrial application prospects.

Description

A 5-aminolevulinic acid producing strain and its construction method and application

Technical Field

The invention relates to the technical field of biotechnology and fermentation engineering, in particular to a 5-aminolevulinic acid producing strain and a construction method and application thereof.

Background technique

5-Aminolevulinic acid (C5O3NH9) is a non-protein amino acid and a common precursor for organisms to synthesize chlorophyll, heme, vitamin B12 and other pyrrole compounds. It is widely present in microorganisms and animal and plant cells and has important applications in medicine, agriculture, health care, chemical industry and other industries.

At present, the synthesis methods of 5-aminolevulinic acid are mainly chemical synthesis and microbial fermentation. The chemical synthesis method has the problems of large environmental pollution and low conversion rate. This method uses a large amount of acid-base reagents and organic reagents, and has the problems of difficult extraction, serious pollution, and high price, which cannot meet the needs of production. The microbial fermentation method uses glucose as the energy source for microbial growth. However, at present, the synthesis of 5-aminolevulinic acid by microorganisms using glucose as a substrate requires the addition of intermediate substances, high production cost, long fermentation cycle, low sugar conversion efficiency, and low yield, which is not conducive to industrial production. Therefore, it is of great value to develop a genetically engineered bacterium that does not add intermediate substances and uses glucose as a substrate to produce 5-aminolevulinic acid cheaply and efficiently.

Summary of the invention

The technical problem to be solved by the present invention is to provide a 5-aminolevulinic acid producing strain.

Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned 5-aminolevulinic acid producing strain.

Another technical problem to be solved by the present invention is to provide the application of the above-mentioned 5-aminolevulinic acid producing strain.

In order to solve the above technical problems, the technical solution of the present invention is:

A plasmid is a plasmid pETJAL, which is transformed from a plasmid pET28a and carries aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE genes and an alaS gene derived from Rhodobactersphaeroides . The nucleotide sequence of the plasmid pET28a is shown in SEQ ID NO.10 in the sequence table.

Preferably, the above-mentioned plasmid carries plasmid elements such as a replication start site, a kanamycin resistance gene, a T7 promoter, and a terminator, and also carries aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE genes derived from E. coli W3110 (E. coli W3110 ATCC 27325) and an alaS gene derived from Rhodobacters phaeroides , and the T7 promoter is used to enhance transcription.

The construction method of the above plasmid uses the ClonExpress® rapid cloning technology of Nanjing Novogene Biotech Co., Ltd. to construct the overexpression plasmid:

(1) Select the cloning site, use the inverse PCR technique to amplify the pET28a linear vector, and use the ClonExpress® recombinase to connect the pET28a linear vector with the cloned fragment T7-aspC to obtain a new plasmid pET-1;

(2) Using the same plasmid construction method, the thrA ^fbr , thrB , and thrC genes driven by the T7 promoter were constructed and expressed in the plasmid pET-1 to obtain pET-2;

(3) Using the same plasmid construction method, the asd gene driven by T7 promoter was constructed into plasmid pET-2 to express pET-3;

(4) Using the same plasmid construction method, the tdh gene driven by the T7 promoter was constructed and expressed in the plasmid pET-3 to obtain pET-4;

(5) The same plasmid construction method was used to construct the itaE gene driven by the T7 promoter into the plasmid pET-4 to obtain pET-5;

(6) The same plasmid construction method was used to construct the alaS gene driven by the T7 promoter and expressed in the plasmid pET-5 to obtain pETJAL.

A 5-aminolevulinic acid producing strain is strain JAL11, which up-regulates aceF, pykF, icd, gdhA, gltx, hemA, heml, and rhtA genes , down-regulates hemB gene through BBa_J23113 promoter, heterologously expresses NCU05151 gene from Neurospora crassa , and carries the above-mentioned high-copy plasmid pETJAL, on which aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE genes of E.coli W3110 and alaS gene from Rhodobacter sphaeroides are expressed.

Preferably, the above-mentioned 5-aminolevulinic acid producing strain uses E.coli W3110 as the chassis strain, uses the BBa_J23113 promoter to down-regulate the hemB gene and integrate it into the ygaY gene site of the genome; uses the trc promoter to strengthen the aceF gene and integrate it into the ylbE gene site of the genome; uses the trc promoter to strengthen the pykF gene and integrate it into the gapC gene site of the genome; uses the trc promoter to strengthen the icd gene and integrate it into the rph gene site of the genome; uses the trc promoter to strengthen the gdhA gene and integrate it into the yeeL gene site of the genome; uses the trc promoter to strengthen the gltX gene and integrate it into the ycgH gene site of the genome; uses the trc promoter to strengthen the heml gene and integrate it into the yjgX gene site of the genome; uses the trc promoter to strengthen the hemA gene and integrate it into the mbhA gene site of the genome; uses the trc promoter to strengthen the rhtA gene and integrate it into the yeeP gene site of the genome; uses the trc promoter to strengthen the gene derived from Neurospora crassa The NCU05151 gene of OR74A is transcribed and integrated into the ycdN gene locus of the genome.

Preferably, in the above-mentioned 5-aminolevulinic acid producing strain, the E. coli W3110 is E. coli W3110 ATCC27325.

Preferably, in the above-mentioned 5-aminolevulinic acid producing strain, the nucleotide sequence of the aceF gene is shown in SEQ ID NO.1 in the sequence listing, the nucleotide sequence of the pykF gene is shown in SEQ ID NO.2 in the sequence listing, the nucleotide sequence of the icd gene is shown in SEQ ID NO.3 in the sequence listing, the nucleotide sequence of the gdhA gene is shown in SEQ ID NO.4 in the sequence listing, the nucleotide sequence of the gltX gene is shown in SEQ ID NO.5 in the sequence listing, the nucleotide sequence of the hemA gene is shown in SEQ ID NO.6 in the sequence listing, the nucleotide sequence of the hemL gene is shown in SEQ ID NO.7 in the sequence listing, the nucleotide sequence of the rhtA gene is shown in SEQ ID NO.8 in the sequence listing, the nucleotide sequence of the NCU05151 gene is shown in SEQ ID NO.9 in the sequence listing, the nucleotide sequence of the plasmid pET28a is shown in SEQ ID NO.10 in the sequence listing, the nucleotide sequence of the trc promoter is shown in SEQ ID NO.11 in the sequence listing, the nucleotide sequence of the BBa_J23113 promoter is shown in SEQ ID NO.12 in the sequence listing, and the nucleotide sequence of the T7 promoter is shown in SEQ ID NO.13 in the sequence listing. The nucleotide sequence of the aspC gene is shown in SEQ ID NO.13, the nucleotide sequence of the thrA ^fbr gene is shown in SEQ ID NO.14 in the sequence listing, the nucleotide sequence of the thrB gene is shown in SEQ ID NO.15 in the sequence listing, the nucleotide sequence of the thrC gene is shown in SEQ ID NO.17 in the sequence listing, the nucleotide sequence of the asd gene is shown in SEQ ID NO.18 in the sequence listing, the nucleotide sequence of the tdh gene is shown in SEQ ID NO.19 in the sequence listing, the nucleotide sequence of the itaE gene is shown in SEQ ID NO.20 in the sequence listing, the nucleotide sequence of the hemB gene is shown in SEQ ID NO.21 in the sequence listing, and the nucleotide sequence of the alaS gene is shown in SEQ ID NO.22 in the sequence listing.

The above-mentioned method for constructing a 5-aminolevulinic acid producing strain is to carry out directional transformation based on the starting strain E. coli W3110 , and the specific steps are as follows:

(1) Chassis bacteria modification: Strengthening the 5-aminolevulinic acid _C5 pathway

The strong trc promoter was used to drive the expression of aceF , pykF , icd , gdhA , gltx , hemA , hemL and the external transporter rhtA gene, which greatly increased the transcription level of the above genes;

(2) Optimizing the acetyl-CoA supply system: Improving conversion rate

The NCU05151 gene from Neurospora crassa was introduced to efficiently utilize carbon sources to produce a large amount of acetyl-CoA, which flows into the TCA cycle, provides a large amount of precursors for the 5-aminolevulinic acid C ₅ metabolic pathway and the glycine metabolic pathway, and accumulates 5-aminolevulinic acid.

(3) Weakening the catabolism of 5-aminolevulinic acid:

The BBa_J23113 promoter was used to down-regulate the hemB gene, weakening the catabolism of 5-aminolevulinic acid by 45 times compared with wild bacteria, weakening the catabolism of 5-aminolevulinic acid and allowing 5-aminolevulinic acid to accumulate efficiently;

(4) pETJAL plasmid system: opening up the glycine pathway

The pETJAL plasmid carries plasmid elements such as a replication initiation site, a kanamycin resistance gene, a T7 promoter, and a terminator. It also carries aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE genes derived from E. coli W3110 and an alaS gene derived from Rhodobacters phaeroides . The aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE, and alaS genes are all transcribed using a T7 promoter. The constructed pETJAL plasmid is electroporated into the competent cells obtained in step (3) to obtain strain JAL11.

Application of the above 5-aminolevulinic acid producing strain in fermentation production of 5-aminolevulinic acid.

Preferably, the above-mentioned 5-aminolevulinic acid producing strain is used, and the strain is fermented and cultured in a culture medium, and the culture medium includes but is not limited to a carbon source, a nitrogen source, an inorganic salt, vitamins, etc.; the fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen conditions, fermentation pressure, fermentation time, etc.

Preferably, the application of the above-mentioned 5-aminolevulinic acid producing strain comprises the following specific steps:

① Slant culture: inoculate the 5-aminolevulinic acid producing strain on a slant medium and culture at 32-35°C for 12-16 hours. The slant medium is a general LB solid medium.

② Shake flask seed culture: Take the solid slant bacteria and inoculate them into the shake flask medium for fermentation. The culture temperature is 32-35℃, the culture time is 12-20h, the shaker speed is 200-240r/min, and the pH is 6.4-6.7;

③ Shake flask fermentation culture: the fermentation inoculation amount is 15-20%, the culture temperature is 32-35℃, the pH is 6.4-6.7, the culture time is 24-36h, and the shaking speed is 200-240r/min.

Preferably, in the application of the above-mentioned 5-aminolevulinic acid producing strain, the seed culture medium used in the step ② is: 25 g/L glucose, 3.5 g/L yeast, 1.3 g/L peptone, 1 g/L (NH ₄ ) ₂ SO _{4 ,} 2 g/L K ₂ HPO ₄ ·3H ₂ O , 2 g/L MgSO ₄ ·7H ₂ O 2 g/L, 1.5 g/L citric acid, 5 mg/L MnSO ₄ ·H ₂ O, 2 mg/L trace elements, and the rest is water.

Preferably, in the application of the above-mentioned 5-aminolevulinic acid producing strain, the fermentation medium used in step ③ is: 15 g/L glucose, 3 g/L yeast powder, 1 g/L peptone, 2 g/L (NH4) ₂ SO ₄ , 5 g/L K ₂ HPO ₄ ·3H ₂ O, 2 g/L MgSO ₄ ·7H ₂ O, 10 mg/L MnSO ₄ ·H ₂ O, 30 mg/L FeSO ₄ ·7H ₂ O, 2 mg/L trace elements, and the rest is water.

Preferably, in the application of the above-mentioned 5-aminolevulinic acid producing strain, the trace elements are 5 mg/L boric acid, 2.6 CoCl, 2.2 _mg / _{L H2O} , 0.4 mg/L _MnSO4 ·H2O, 0.6 mg/L CuSO4·7H2O _, and _0.6 mg/L _ZnSO4 ·7H2O. The above-mentioned solid components are weighed and dissolved in 1 L of water and stored at 4°C.

The above culture media can be prepared by standard methods.

Beneficial effects:

The 5-aminolevulinic acid producing strain has good 5-aminolevulinic acid synthesis ability, stable performance, high acid production efficiency, up-regulated aceF, pykF, icd, gdhA, gltx, hemA, heml, rhtA genes , down-regulated hemB gene through promoter, heterologously expressed NCU05151 gene derived from Neurospora crassa , and carried plasmid pETJAL at the same time, on which T7 promoter was used to express aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE genes derived from E. coli W3110, and heterologously expressed alaS gene derived from Rhodobacters phaeroides , and the phosphoketolase of the strain was derived from NCU05151 gene of Neurospora crassa, which efficiently produced acetyl-CoA to enter the TCA cycle, reduced carbon source loss, and enhanced C ₅ pathway, and the key enzymes of the glycine pathway are expressed using a highly stable plasmid system, opening up the glycine pathway, allowing the C ₅ metabolic pathway of 5-aminolevulinic acid and the glycine metabolic pathway to play a role in the engineered bacteria at the same time, so that 5-aminolevulinic acid can be efficiently accumulated, which can effectively improve the synthesis efficiency of 5-aminolevulinic acid and the production level of 5-aminolevulinic acid, and realize the efficient production of 5-aminolevulinic acid, which has excellent industrial application prospects. Specifically:

(1) By increasing the transcription level of aceF and pykF genes, acetyl-CoA can be effectively increased to enter the TCA cycle; by increasing the transcription level of icd gene, α-ketoglutarate accumulation can be effectively increased; by increasing the transcription level of gdhA, gltx, heml, and hemA genes, the C5 pathway is enhanced to enable efficient accumulation of 5-aminolevulinic acid; by increasing the transcription level of rhtA gene, the level of 5-aminolevulinic acid transport outside the cell is enhanced;

(2) Using the pETJAL plasmid, the T7 promoter on the plasmid overexpressed the aspC, thrA ^fbr , thrB, thrC, asd, tdh, ltaE genes from E. coli W3110 and the alaS gene from Rhodobacters phaeroides, opening up the glycine pathway and allowing efficient accumulation of 5-aminolevulinic acid;

(3) CRISPR-Cas9 gene editing technology was used to open up the _C5 metabolic pathway of 5-aminolevulinic acid, and the pETJAL plasmid was constructed to open up the glycine metabolic pathway of 5-aminolevulinic acid. The _C5 metabolic pathway and the glycine metabolic pathway functioned simultaneously in the engineered bacteria, resulting in efficient accumulation of 5-aminolevulinic acid.

(4) The NCU05151 gene from Neurospora crassa was introduced. Phosphoketolase efficiently decomposes fructose hexaphosphate into acetyl-CoA, reducing carbon source loss and providing a large amount of precursors for the ₅ -aminolevulinic acid C5 metabolic pathway and the glycine metabolic pathway, thereby achieving the purpose of 5-aminolevulinic acid accumulation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the structure of plasmid pET28a.

Figure 2 is a schematic diagram of the structure of plasmid pETJAL.

FIG. 3 is a diagram showing the process of transforming the de novo synthesis pathway of 5-aminolevulinic acid genetically engineered bacteria.

Detailed ways

In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

The percentage sign "%" involved in the examples, unless otherwise specified, refers to mass percentage. The percentage of a solution refers to the number of grams of a solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

The starting strain used in the examples is wild-type E. coli W3110ATCC 27325 (commercially available), and the corresponding promoters and genes are shown in the sequence table. The primers used in the construction of the strains involved are shown in Table 1.

Table 1 Primers involved in strain construction

Primer name serial number Primer sequence (5'-3') pGRB-ygaY-S SEQ ID NO.23 AGTCCTAGGTATAATACTAGTCTCAACTACCCACAGTTGTTGTTTTAGAGCTAGAA pGRB-ygaY-A SEQ ID NO.24 TTCTAGCTCTAAAACAACAACTGTGGGTAGTTGAGACTAGTATTATACCTAGGACT ygaY-US SEQ ID NO.25 CCTACAAACCACATCGCACATT ygaY-UA SEQ ID NO.26 GCTAGCACAGTCCCTAGGACTGAGCTAGCTGTAAAACACCGAAGCAACCCAAAAGA ygaY-DS SEQ ID NO.27 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACCTTTTA ygaY-DA SEQ ID NO.28 GGAGTAGGGCTTTCCATAGAGTGT hemB-DS SEQ ID NO.29 CTAGCTCAGTCCTAGGGACTGTGCTAGCAGGAAACAGACCATGACAGACTTAATCCAACGCCCT HemB-DA SEQ ID NO.30 AACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAACGCAGAATCTTCTTCTCAGCCAAAT pGRB-ylbE-S SEQ ID NO.31 AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA pGRB-ylbE-A SEQ ID NO.32 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT ylbE-US SEQ ID NO.33 ACCCAACCTTACGCAACCAGTTAA YLbE-UA SEQ ID NO.34 ATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCATTG ylbE-DS SEQ ID NO.35 ACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAACA ylbE-DA SEQ ID NO.36 GGGCGTAACTCAGCAGGCAG aceF-S SEQ ID NO.37 TGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGCTATCGAAATCAAAGTACCG aceF-A SEQ ID NO.38 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACATCACCAGACGGCGAATGT pGRB-gapC-S SEQ ID NO.39 AGTCCTAGGTATAATACTAGTAGGTCATGCGCAACGCGTGCGTTTTAGAGCTAGAA pGRB-gapC-A SEQ ID NO.40 TTCTAGCTCTAAAACGCACGCGTTGCGCATGACCTACTAGTATTATACCTAGGACT gapC-US SEQ ID NO.41 TGGGAAGAAACCACGAAACTCCA gapC-UA SEQ ID NO.42 TATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGTTTCAGCAGGTAGGCGAGAA gapC-DS SEQ ID NO.43 TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAAACGGTCGCCTGGTACG gapC-DA SEQ ID NO.44 TTATCCGCCGACATTGCTGC pykF-S SEQ ID NO.45 TGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAAAGACCAAAATTGTTTGCACC pykF-A SEQ ID NO.46 ACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGGACGTGAACAGATGCGG pGRB-rph-S SEQ ID NO.47 AGTCCTAGGTATAATACTAGTTGCGACGTGCTTCAGGCTGAGTTTTAGAGCTAGAA pGRB-rph-A SEQ ID NO.48 TTCTAGCTCTAAAACTCAGCCTGAAGCACGTCGCAACTAGTATTATACCTAGGACT rph-US SEQ ID NO.49 ATAGCGCAGGGTACATTCCACT rph-UA SEQ ID NO.50 TTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACAC rph-DS SEQ ID NO.51 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGCCGCAGAGACCGACATGAA rph-DA SEQ ID NO.52 ACAGCGGTTGTGGTGGCAAT icd-S SEQ ID NO.53 TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAAAGTAAAGTAGTTGTTCCGGC icd-A SEQ ID NO.54 AAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACATGTTTTCGATGATCGCGTCAC pGRB-yeeL-S SEQ ID NO.55 AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA pGRB-yeeL-A SEQ ID NO.56 TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT yeeL-US SEQ ID NO.57 TTCATCGGGACGAGTGGAGA yeeL-UA SEQ ID NO.58 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCATAGCATCGCCAATCTGA yeeL-DS SEQ ID NO.59 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATAAAGCC yeeL-DA SEQ ID NO.60 CATTCCCTCTACAGAACTAGCCCTT gdhA-S SEQ ID NO.61 GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGATCAGACATATTCTCTGGAGTC gdhA-A SEQ ID NO.62 GATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAAATCACACCCTGCGCCAG pGRB-ycgh-S SEQ ID NO.63 AGTCCTAGGTATAATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA pGRB-ycgh-A SEQ ID NO.64 TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT ycgh-US SEQ ID NO.65 TAAACTCGTCAGCGGCACAAC ycgh-UA SEQ ID NO.66 ATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTAGGCGTTTCTGTTGATTCTGAA ycgh-DS SEQ ID NO.67 TTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGTGTCGGATTATCGTTCGA ycgh-DA SEQ ID NO.68 GATTCAGGTTGCCATTTACGCCA gltX-S SEQ ID NO.69 ATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAATCAAAACTCGCTTCGCG gltX-A SEQ ID NO.70 TTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACTGCTGATTTTCGCGTTCAGC pGRB-yjgX-S SEQ ID NO.71 AGTCCTAGGTATAATACTAGTTCGCGACCACCGTAACTGGCGTTTTAGAGCTAGAA pGRB-yjgX-A SEQ ID NO.72 TTCTAGCTCTAAAACGCCAGTTACGGTGGTCGCGAACTAGTATTATACCTAGGAC yjgX-US SEQ ID NO.73 GGAAGTCAACGGGTTATGCGG yjX-UA SEQ ID NO.74 TTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAATCACCACGAATACCAGAATCGC yjgX-DS SEQ ID NO.75 ACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACAGTGTCTTCCCTGAGCCG xyj SEQ ID NO.76 GGCGAAGGATACCATCAAGCTG hemL-S SEQ ID NO.77 GGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGTAAGTCTGAAAATCTTTACAGCGC hemL-A SEQ ID NO.78 AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCACAACTTCGCAAACACCCG pGRB-mbhA-S SEQ ID NO.79 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACACGGTGGCAGGTTTTGG pGRB-mbhA-A SEQ ID NO.80 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-US SEQ ID NO.81 GCCAGCACGAACATAATCCC mbhA-UA SEQ ID NO.82 GGTCTGTTTCCTGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAACACGGTGGCAGGTTTTGG mbhA-DS SEQ ID NO.83 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-DA SEQ ID NO.84 CGGCGTAATCACAAACTGGC hemA-S SEQ ID NO.85 ATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACCCTTTTAGCACTCGGTATCAA hemA-A SEQ ID NO.86 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTACTCCAGCCCGAGGCT pGRB-yeeP-S SEQ ID NO.87 AGTCCTAGGTATAATACTAGTTCCCCTGATGACCCGACTGCGTTTTAGAGCTAGAA pGRB-yeeP-A SEQ ID NO.88 TTCTAGCTCTAAAACGCAGTCGGGTCATCAGGGGAACTAGTATTATACCTAGGACT yeeP-US SEQ ID NO.89 GGTCAGGAGGTAACTTATCAGCG yeeP-UA SEQ ID NO.90 CCACACATTATACGAGCCGGATGATTAATTGTCAAATGGCAGGGCTCCGTTTT yeeP-DS SEQ ID NO.91 CGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAACTGGATTTTCTTCTGAACCTGT yeeP-DA SEQ ID NO.92 ACGATGTCAGCAGCCAGCA rhtA-S SEQ ID NO.93 CATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCCTGGTTCATTACGTAAAATGCCG rhtA-A SEQ ID NO.94 AAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAATTAATGTCTAATTCTTTTATTTTGCTCTCTTTGCGTACTG pGRB-ycdN-S SEQ ID NO.95 AGTCCTAGGTATAATACTAGTGCGTGGAAATCATCATGGCTGTTTTAGAGCTAGAA pGRB-ycdN-A SEQ ID NO.96 TTCTAGCTCTAAAACAGCCATGATGATTTCCACGCACTAGTATTATACCTAGGACT ycdN-US SEQ ID NO.97 GATTTTGACGCCACCAACACC YcD SEQ ID NO.98 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAATCCACATCACACAATCCATC ycdN-DS SEQ ID NO.99 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAAGGGATTTTTGGCTATCAGG YcdN-DA SEQ ID NO.100 GTATTCGCCAGGCTGTAAATTC Ncu05151-S SEQ ID NO.101 TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGGCGGAACACAGATCAC Ncu05151-A SEQ ID NO.102 ACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTACTCGAACTTGGGCAGTTGG aspC-pet-S SEQ ID NO.103 GCCCTGTAATACGACTCACTATAGGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGTTTGAGAACATTACCGCCGC aspC-pet-A SEQ ID NO.104 CGGGAGCAGACAAGCCCGTCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTACAGCACTGCCACAATCGC thrA ^fbr -pet-S SEQ ID NO.105 ATAATGGTTAATACGACTCACTATAGGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGCGAGTGTTGAAGTTCGGC thrC-pet-A SEQ ID NO.106 AGCGCCCTGCCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTACTGATGATTCATCATCAATTTACGCAACG asd-pet-S SEQ ID NO.107 TCTAATACGACTCACTATAGGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAAAAATGTTGGTTTTATCGGCTGG asd-pet-A SEQ ID NO.108 CCACCAAACGTTTCGGCGACAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTACGCCAGTTGACGAAGCATCC tdh-pet-S SEQ ID NO.109 CGCTAATACGACTCACTATAGGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAAAGCGTTATCCAAACTGAAAGC tdh-pet-A SEQ ID NO.110 GCGTCGAGATCCCGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTAATCCCAGCTCAGAATAACTTTCCCG ltaE-pet-S SEQ ID NO.111 CGTTAATACGACTCACTATAGGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGATTGATTTACGCAGTGATACCGT ltaE-pet-A SEQ ID NO.112 TCCTTGCGGCGGCGGTGCTCACAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTAACGCGCCAGGAATGCAC alaS-pet-S SEQ ID NO.113 ACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCATGCCGTCCTTGAACGATATCCG alaS-pet-A SEQ ID NO.114 CTCAGTGGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTCAGCCTCCCATCACCG

As shown in FIG3 , a 5-aminolevulinic acid producing strain was constructed by the following four modules:

(1) Chassis bacteria transformation:

Increase the transcription level of the aceF gene, increase the transcription level of the pykF gene, increase the transcription level of the icd gene, increase the transcription level of the gdhA gene, increase the transcription level of the gltx gene, increase the transcription level of the hemA gene, increase the transcription level of the heML gene, and increase the transcription level of the external transport protein rhtA gene.

(2) Optimizing the acetyl-CoA supply system:

The NCU05151 gene from Neurospora crassa was introduced, which can efficiently utilize carbon sources to produce a large amount of acetyl-CoA, which flows into the TCA cycle, providing a large amount of precursors for the ₅ -aminolevulinic acid C5 metabolic pathway and the glycine metabolic pathway, thereby accumulating 5-aminolevulinic acid.

(3) Weakening the catabolism of 5-aminolevulinic acid:

The BBa_J23113 promoter was used to downregulate the hemB gene, weakening the catabolism of 5-aminolevulinic acid and causing its accumulation.

(4) pETJAL plasmid (as shown in FIG. 2 , it is particularly noted that, except for the plasmid elements specifically mentioned in the present invention, the other elements shown in the diagram can be replaced by any currently known elements with equivalent effects) system:

The pETJAL plasmid is transformed from the plasmid pET28a (as shown in FIG. 1 ), and carries plasmid elements such as a replication initiation site, a kanamycin resistance gene, a T7 promoter, and a terminator. It also carries aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE genes derived from E. coli W3110 and an alaS gene derived from Rhodobacters phaeroides . The aspC, thrA ^fbr , thrB, thrC, asd, tdh, itaE, and alaS genes are all transcribed using a T7 promoter.

The gene editing method used in the above gene manipulation is referred to in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.). The professional terms involved in the present invention can be explained in the article unless otherwise noted. "Introduction" refers to the insertion of exogenous genes into the genome of engineered bacteria after connecting them to promoters and terminators.

Example 1

This example is intended to illustrate the specific construction steps of strain JAL11. In particular, if there is a method for the same type of gene manipulation in the example, it will only be provided once and annotated without further elaboration.

① Use BBa_J23113 promoter to downregulate hemB gene at the ygaY pseudogene site: Using E.coli W3110 genome as template, ygaY-US, ygaY-UA and ygaY-DS, ygaY-DA were used to obtain upstream and downstream homology arms by PCR amplification, then, using the upstream and downstream homology arms as templates and hemB -S and hemB -A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-ygaY-s and pGRB-ygaY-a were used as primers to anneal the gRNA fragments, which were connected with the pGRB vector to obtain ygaY-pGRB; E.coli W3110 electroporation competent cells were prepared, the overlapping fragments and ygaY-pGRB were electroporated into the competent cells, and positive transformants were screened to obtain strain JAL1.

②Use trc promoter to control overexpression of aceF gene at the ylbE pseudogene site: Using E.coli W3110 genome as template, ylbE-US, ylbE-UA and ylbE-DS, ylbE-DA were used to obtain upstream and downstream homology arms by PCR amplification, then, using upstream and downstream homology arms as templates and aceF-S and aceF-A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-ylbE-s and pGRB-ylbE-a were used as primers to anneal gRNA fragments, which were connected with pGRB vector to obtain ylbE-pGRB; E.coli W3110 electroporation competent cells were prepared, the overlapping fragments and ylbE-pGRB were electroporated into competent cells, and positive transformants were screened to obtain strain JAL2.

③ Use trc promoter to control overexpression of pykF gene at gapC pseudogene site: Using E.coli W3110 genome as template, gapC-US, gapC-UA and gapC-DS, gapC-DA were used to obtain upstream and downstream homology arms by PCR amplification, then, using upstream and downstream homology arms as templates and pykF-S and pykF-A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-gapC-s and pGRB-gapC-a were used as primers to anneal gRNA fragments, which were connected with pGRB vector to obtain gapC-pGRB; E.coli W3110 electroporation competent cells were prepared, the overlapping fragments and gapC-pGRB were electroporated into competent cells, and positive transformants were screened to obtain strain JAL3.

④ Use trc promoter to control icd gene overexpression at rph pseudogene site: Using E.coli W3110 genome as template, rph-US, rph-UA and rph-DS, rph-DA were used to obtain upstream and downstream homology arms by PCR amplification, then, using upstream and downstream homology arms as templates and icd-S and icd-A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-rph-s and pGRB-rph-a were used as primers to anneal gRNA fragments, and they were connected with pGRB vector to obtain rph-pGRB; E.coli W3110 electroporation competent cells were prepared, the overlapping fragments and rph-pGRB were electroporated into competent cells, and positive transformants were screened to obtain strain JAL4.

⑤ Use trc promoter to control gdhA gene overexpression at the yeeL pseudogene site: Using E.coli W3110 genome as template, yeeL-US, yeeL-UA and yeeL-DS, yeeL-DA were used to obtain upstream and downstream homology arms by PCR amplification, then, using the upstream and downstream homology arms as templates and gdhA-S and gdhA-A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-yeeL-s and pGRB-yeeL-a were used as primers to anneal the gRNA fragments, and they were connected with the pGRB vector to obtain yeeL-pGRB; E.coli W3110 electroporation competent cells were prepared, the overlapping fragments and yeeL-pGRB were electroporated into the competent cells, and the positive transformants were screened to obtain strain JAL5.

⑥ Use trc promoter to control gltX gene overexpression at ychH pseudogene site: Using E.coli W3110 genome as template, ychH-US, ychH-UA and ychH-DS, ychH-DA were used to obtain upstream and downstream homology arms by PCR amplification, then, using upstream and downstream homology arms as templates and gltX-S and gltX-A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-ychH-s and pGRB-ychH-a were used as primers to anneal gRNA fragments, which were connected with pGRB vector to obtain ychH-pGRB; E.coli W3110 electroporation competent cells were prepared, the overlapping fragments and ychH-pGRB were electroporated into competent cells, and positive transformants were screened to obtain strain JAL6.

⑦ Use trc promoter to control heml gene overexpression at the yjgX pseudogene site: Using E.coli W3110 genome as template, yjgX-US, yjgX-UA and yjgX-DS, yjgX-DA were used to obtain upstream and downstream homology arms by PCR amplification, then, using upstream and downstream homology arms as templates and heml-S and heml-A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-yjgX-s and pGRB-yjgX-a were used as primers to anneal the gRNA fragments, which were connected with the pGRB vector to obtain yjgX-pGRB; E.coli W3110 electroporation competent cells were prepared, the overlapping fragments and yjgX-pGRB were electroporated into the competent cells, and the positive transformants were screened to obtain strain JAL7.

⑧Use trc promoter to control overexpression of hemA gene at mbhA pseudogene site: Using E.coli W3110 genome as template, mbhA-US, mbhA-UA and mbhA-DS, mbhA-DA were used to obtain upstream and downstream homology arms by PCR amplification, then, using upstream and downstream homology arms as templates and hemA-S and hemA-A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-mbhA-s and pGRB-mbhA-a were used as primers to anneal gRNA fragments, and they were connected with pGRB vector to obtain mbhA-pGRB; E.coli W3110 electroporation competent cells were prepared, the overlapping fragments and mbhA-pGRB were electroporated into competent cells, and positive transformants were screened to obtain strain JAL8.

⑨ Use the trc promoter to control the overexpression of the rhtA gene at the yeeP pseudogene site: Using the E.coli W3110 genome as a template, yeeP-US, yeeP-UA and yeeP-DS, yeeP-DA were used to obtain the upstream and downstream homology arms by PCR amplification, and then, using the upstream and downstream homology arms as templates and rhtA-S and rhtA-A as primers, overlapping fragments were amplified by overlapping PCR; using pGRB-yeeP-s and pGRB-yeeP-a as primers, annealing obtained the gRNA fragment, and connected it with the pGRB vector to obtain yeeP-pGRB; prepare E.coli W3110 electroporation competent cells, electroporate the overlapping fragments together with yeeP-pGRB into the competent cells, and screen to obtain positive transformants to obtain strain JAL9.

⑩ Use the trc promoter to control the overexpression of the NCU05151 gene at the ycdN pseudogene site: Using the E. coli W3110 genome as a template, ycdN-US, ycdN-UA and ycdN-DS, ycdN-DA were used to obtain the upstream and downstream homology arms by PCR amplification, then, using the upstream and downstream homology arms as templates and NCU05151-S and NCU05151-A as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-ycdN-S and pGRB-ycdN-A were used as primers to anneal the gRNA fragments, and they were connected with the pGRB vector to obtain ycdN-pGRB; E. coli W3110 electroporation competent cells were prepared, the overlapping fragments and ycdN-pGRB were electroporated into the competent cells, and the positive transformants were screened to obtain the strain JAL10.

⑪Transform the plasmid pETJAL to obtain the engineered bacterium JAL11: Transform the complete plasmid pETJAL into JAL10 competent cells using electroporation (or other methods such as chemical transformation) to obtain the engineered bacterium JAL11.

Example 2

This example is intended to illustrate the construction method of plasmid pETJAL in Example 1, and the specific steps are as follows:

Using the E.coliW3110 ATCC 27325 genome as a template and aspC-pet-S and aspC-pet-A as primers, the aspC gene was amplified; using ^thrAFbr -pet-S and thrC-pet-A as primers, the ^thrAFbr , thrB and thrC genes were amplified; using asd-pet-S and asd-pet-A as primers, the asd gene was amplified; using tdh-pet-S and tdh-pet-A as primers, the tdh gene was amplified; using itaE-pet-S and itaE-pet-A as primers, the itaE gene was amplified; using the Rhodobactersphaeroides genome as a template and alaS-pet-S and alaS-pet-A as primers, the alaS gene was amplified. The above-mentioned genes were overexpressed in the plasmid via the T7 promoter.

The gene fragment and the plasmid linearized vector are connected by recombinase to obtain a complete plasmid. Any recombinase can be used for the connection method. In this embodiment, the ClonExpress® rapid cloning technology of Nanjing Novogene Biotech Co., Ltd. is used to construct the overexpression plasmid.

Example 3

This example is intended to illustrate the application of shake flask fermentation of the engineered bacteria JAL11. The specific steps are as follows:

① Slant culture: Take the bacteria stored in a -80℃ refrigerator and inoculate it on the slant culture medium, and culture it at 32-35℃ for 14h. The slant culture medium should be the universal LB solid culture medium.

② Shake flask seed culture: Take the solid slant bacteria and inoculate it into the shake flask medium for fermentation. The culture temperature is 36-37℃, the culture time is 16h, the shaker speed is 220r/min, the pH is 6.4-6.7, and the seed culture medium is 25 g/L glucose, 3.5 g/L yeast, 1.3 g/L peptone, 1 g/L (NH ₄ ) ₂ SO ₄ , 2 g/L K ₂ HPO ₄ ·3H ₂ O, 2 g/L MgSO ₄ ·7H ₂ O, 1.5 g/L citric acid, 5 mg/L MnSO ₄ ·H ₂ O, 2 mg/L trace elements, and the rest is water.

③ Shake flask fermentation culture: the fermentation inoculation amount is 15-20%, the culture temperature is 32-35℃, the pH is 6.4-6.7, the culture time is 30h, and the shaker speed is 220r/min; the fermentation medium used is: glucose 15 g/L, yeast powder 3 g/L, peptone 1g/L, (NH4) ₂ SO ₄ 2 g/L, K ₂ HPO ₄ ·3H ₂ O 5 g/L, MgSO ₄ ·7H ₂ O 2 g/L, MnSO ₄ ·H ₂ O 10 mg/L, FeSO ₄ ·7H ₂ O 30 mg/L, trace elements 2 mg/L, and the rest is water.

Using wild-type E. coli W3110 as the control group, after 36 hours of fermentation verification, the wild-type E. coli W3110 failed to accumulate 5-aminolevulinic acid, while the engineered bacteria JAL11 described in Example 1 accumulated 9.3 g/L 5-aminolevulinic acid, proving the effectiveness of the strain.

Example 4

This example aims to illustrate the effect of the combined action of the glycine pathway and the _C5 pathway and the _C5 pathway alone on the production capacity of 5-aminolevulinic acid in strains JAL11 (group A) and JAL10 (group B) under the same culture conditions. The experiment is divided into two groups, A and B, in which:

Group A and Group B adopted the same culture method as in Example 3. The difference between Group B and Group A was that pETJAL plasmid was present in the E. coli of Group A. The fermentation was carried out for a total of 36 hours, and samples were taken at 12 hours, 24 hours, and 36 hours to detect the 5-aminolevulinic acid yield and biomass parameters, respectively. The results are shown in Table 2 below.

Table 2 5-aminolevulinic acid yield and biomass parameters

12h (Group A/Group B) 24h (Group A/Group B) 36h (Group A/Group B) A/B5-aminolevulinic acid (g/L) 1.4/1.1 6.2/4.5 9.3/7.2 A/B biomass (OD600 _nm ) 11.2/10.3 24.3/20.1 32.2/28.6

The results showed that the production of 5-aminolevulinic acid by the combined action of glycine pathway and _C5 pathway was higher than that by the _C5 pathway alone. Therefore, the rational application of the combined action of glycine pathway and _C5 pathway accumulated 9.3 g/L 5-aminolevulinic acid, which proved the effectiveness of the strain.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims (6)

1. A strain producing 5-aminolevulinic acid, characterized by: using E.coli W3110 ATCC 27325 as the chassis strain, the aceF, pykF, icd, gdhA, gltx, hemA, hemL, rhtA gene, in particular, The aceF gene is enhanced by the trc promoter and integrated at the genomic ylbE gene locus, the pykF gene is enhanced by the trc promoter and integrated at the genomic gapC gene locus, the icd gene is enhanced by the trc promoter and integrated at the genomic rph gene locus, the gdhA gene is enhanced by the trc promoter and integrated at the genomic yeeL gene locus, the gltX gene is enhanced by the trc promoter and integrated at the genomic ycgH gene locus, the hemL gene is enhanced by the trc promoter and integrated at the genomic yjgX gene locus, the hemA gene is enhanced by the trc promoter and integrated at the genomic mbhA gene locus, the rhtA gene is enhanced by the trc promoter and integrated at the genomic yeeP gene locus, the hemB gene is down-regulated by the BBa_J23113 promoter, And integrated at the genome ygaY locus, the trc promoter is used to strengthen the NCU05151 gene from Neurospora crassa and integrated at the genome ycdN locus, and at the same time carries plasmid pETJAL, the plasmid pETJAL is modified by plasmid pET28a and carries aspC, thrA ^fbr, thrB, thrC, asd, tdh, itaE genes and alaS gene from Rhodobactersphaeroides, The nucleotide sequence of the plasmid pET28a is shown in a sequence table SEQ ID NO.10, the nucleotide sequence of the aspC gene is shown in a sequence table SEQ ID NO.14, the nucleotide sequence of the thrA ^fbr gene is shown in a sequence table SEQ ID NO.15, the nucleotide sequence of the thrB gene is shown in a sequence table SEQ ID NO.16, the nucleotide sequence of the thrC gene is shown in a sequence table SEQ ID NO.17, the nucleotide sequence of the asd gene is shown in a sequence table SEQ ID NO.18, the nucleotide sequence of the tdh gene is shown in a sequence table SEQ ID NO.19, the nucleotide sequence of the itaE gene is shown in a sequence table SEQ ID NO.20, and the nucleotide sequence of the alaS gene is shown in a sequence table SEQ ID NO. 22; The nucleotide sequence of the aceF gene is shown in a sequence table SEQ ID NO.1, the nucleotide sequence of the pykF gene is shown in a sequence table SEQ ID NO.2, the nucleotide sequence of the icd gene is shown in a sequence table SEQ ID NO.3, the nucleotide sequence of the gdhA gene is shown in a sequence table SEQ ID NO.4, the nucleotide sequence of the gltX gene is shown in a sequence table SEQ ID NO.5, the nucleotide sequence of the hemA gene is shown in a sequence table SEQ ID NO.6, the nucleotide sequence of the hemL gene is shown in a sequence table SEQ ID NO.7, The nucleotide sequence of rhtA gene is shown in a sequence table SEQ ID NO.8, the nucleotide sequence of NCU05151 gene is shown in a sequence table SEQ ID NO.9, the nucleotide sequence of trc promoter is shown in a sequence table SEQ ID NO.11, the nucleotide sequence of BBa_J23113 promoter is shown in a sequence table SEQ ID NO.12, and the nucleotide sequence of hemB gene is shown in a sequence table SEQ ID NO. 21.

2. The method for constructing a strain producing 5-aminolevulinic acid as defined in claim 1, characterized by: the method is characterized by carrying out directional transformation on the basis of an original strain E.coli W3110, and comprises the following specific steps:

(1) And (3) modifying chassis bacteria: the trc strong promoter is used for respectively promoting expression aceF, pykF, icd, gdhA, gltx, hemA, hemL and the outer transport protein rhtA gene;

(2) Optimizing the acetyl-CoA supply system: introducing an NCU05151 gene derived from Neurospora crassa;

(3) Attenuation of 5-aminolevulinic acid catabolism: down-regulating hemB gene with BBa_J23113 promoter;

(4) pETJAL plasmid System: and (3) transforming the plasmid pETJAL into the competent cells obtained in the step (3) to obtain the recombinant DNA.

3. Use of the 5-aminolevulinic acid-producing strain according to claim 1 for the fermentative production of 5-aminolevulinic acid.

4. Use of a strain producing 5-aminolevulinic acid according to claim 3, characterized in that: the method comprises the following specific steps:

① Slant culture: inoculating the strain on a slant culture medium, and culturing at 32-35 ℃ for 12-16h;

② Shake flask seed culture: inoculating the strain into shake flask culture medium for fermentation at 32-35deg.C for 12-20 hr at rotation speed of 200-240r/min and pH of 6.4-6.7;

③ Shake flask fermentation culture: the fermentation inoculation amount is 15-20%, the culture temperature is 32-35 ℃, the pH is 6.4-6.7, the culture time is 24-36h, and the rotation speed of a shaking table is 200-240r/min.

5. The use of a strain producing 5-aminolevulinic acid according to claim 4, characterized in that: the seed culture medium used in step ② is: glucose 25 g/L, yeast 3.5 g/L, peptone 1.3g/L,(NH₄)₂SO₄ 1 g/L,K₂HPO₄·3H₂O 2 g/L,MgSO₄·7H₂O 2 g/L, citric acid 1.5 g/L, mnSO ₄·H₂ O5 mg/L, trace elements 2 mg/L and the balance of water; the fermentation medium adopted in the step ③ is as follows: 15 g/L of glucose, 3 g/L of yeast powder, 2 mg/L of peptone 1 g/L,(NH4)₂SO₄ 2 g/L,K₂HPO₄·3H₂O 5 g/L,MgSO₄·7H₂O 2 g/L,MnSO₄·H₂O 10 mg/L,FeSO₄·7H₂O 30 mg/L, trace elements and the balance of water.

6. The use of a strain producing 5-aminolevulinic acid according to claim 5, characterized in that: the microelements are boric acid 5mg/L,CoCl2.6,H₂O 2.2mg/L,MnSO₄·H₂O 0.4mg/L,CuSO₄·7H₂O 0.6mg/L,ZnSO₄·7H₂O 0.6mg/L.