CN117802022A - Genetic engineering bacteria for producing tetrahydropyrimidine, construction method and application thereof - Google Patents

Abstract

The invention belongs to the technical fields of genetic engineering and fermentation engineering, and specifically relates to a genetically engineered bacterium that produces ectoine and its construction method and application. The present invention uses Escherichia coli BL21 (DE3) as the starting strain to overexpress gene cluster ectABC, aspartate kinase gene lysC and aspartate ammonia lyase gene aspA, and for the first time uses growth-dependent promoters fliA, fliC, and flgC to respectively regulate the expression of diaminopimelate decarboxylase gene lysA and homoserine dehydrogenase gene metL. This combination of regulation achieves heterologous expression of ectoine without affecting the production of key amino acids to meet the bacterial requirements. While growing, the yield of ectoine was effectively increased.

Description

A genetically engineered bacterium producing ectoine and its construction method and application

Technical field

The invention belongs to the technical fields of genetic engineering and fermentation engineering, and specifically relates to a genetically engineered bacterium that produces ectoine and its construction method and application.

Background technique

There are two main methods for the synthesis of ectoine: chemical synthesis and biological synthesis. There are many problems with chemical synthesis methods, including complex production processes, low synthesis yields, many by-products with similar chemical properties to the target product, and high difficulty in downstream separation and purification. The biosynthetic method mainly uses enzyme catalysis and fermentation. In the biological fermentation method, it is first necessary to construct the biological metabolic pathway of ectoine in the bacteria. Through the metabolic branch pathway of aspartic acid, the synthesis process of ectoine It can be completed in three steps. First, aspartate kinase (LysC) catalyzes aspartate to generate β-aspartyl phosphate; then, it is catalyzed by aspartate semialdehyde dehydrogenase (AsD) to generate aspartate- β-semialdehyde; finally, the synthesis of ectoine is completed under the catalysis of three enzymes encoded by ectABC.

In existing studies, the metabolic pathway of ectoine in Escherichia coli is usually constructed by strengthening the strength of the target product pathway, while the branch byproduct pathway is knocked out to ensure the carbon flow of the target product. This approach is reasonable at the design level, but it may have a negative impact on bacterial growth. It is very likely that the branch pathway product is beneficial to bacterial growth, and once knocked out, it will affect bacterial growth. In the production strain of ectoine, the carbon flow to ectoine is reduced due to the presence of branch byproducts lysine and homoserine. According to previous research experience, knocking out the diaminopimelate decarboxylase gene lysA and the homoserine dehydrogenase gene metL is helpful to increase the yield, but it is not conducive to bacterial growth. The lack of key branch pathways leads to amino acid deficiency, which will inhibit bacterial growth to a certain extent, causing certain pressure on the bacteria, and thus affecting the production of ectoine.

Summary of the invention

The object of the present invention is to overcome the shortcomings of the existing technology and provide a genetically engineered bacterium that produces ectoine, so as to achieve a more reasonable growth state of the bacterial cells and effectively increase the yield of ectoine.

In order to achieve the above objectives, the technical solutions adopted by the present invention include:

In a first aspect, the present invention provides an ectoine-producing Escherichia coli engineering strain, which uses a growth-dependent promoter to regulate the diaminopimelate decarboxylase gene lysA and/or homoserine dehydrogenation respectively. Expression of enzyme gene metL; the E. coli engineered bacterium overexpresses recombinant genes, and the recombinant genes include gene cluster ectABC.

The present invention utilizes synthetic biology technology and genetic engineering methods, uses Escherichia coli as the starting strain, expresses the gene cluster ectABC related to the ectoine synthesis pathway derived from Halomonas elongata, and realizes the heterologous synthesis of ectoine; at the same time, it replaces The competition pathway metabolizes the promoters of diaminopimelate decarboxylase lysA and homoserine dehydrogenase metL, making the growth state of the bacteria more reasonable, thereby effectively increasing the production of ectoine.

Preferably, the growth-dependent promoter includes at least one of fliA, fliC, and flgC promoters; the nucleotide sequence of the fliA promoter is shown in SEQ ID NO: 1, and the nucleotide sequence of the fliC promoter is The sequence is shown in SEQ ID NO: 2, and the nucleotide sequence of the flgC promoter is shown in SEQ ID NO: 3.

Preferably, the growth-dependent promoter includes at least one of fliC and flgC promoters.

The present invention uses a growth-dependent promoter to replace the original promoters of the diaminopimelate decarboxylase gene lysA and the homoserine dehydrogenase gene metL, thereby regulating the production of homoserine and lysine in the competitive branch to achieve bacterial growth. The production of key amino acids is not affected during the growth stage of the cell, satisfying the growth of the cell. At the same time, the expression of amino acids is reduced during the stable phase of the cell, and the carbon flow is concentrated on the expression path of ectoine, thereby achieving the purpose of high-yield ectoine. Experimental research found that the fliA, fliC, and flgC promoters can effectively increase the ectoine production of the engineered strain when regulating the expression of lysA and metL respectively. When the fliC or flgC promoter is used to control the ectoine production, the production improvement effect of ectoine is most significant.

Preferably, the recombinant gene further includes aspartokinase gene lysC and aspartate ammonia lyase gene aspA.

On the basis of the overexpressed gene cluster ectABC, the present invention further improves ectoine by combining the expression of the aspartate kinase gene lysC derived from Corynebacterium glutamicum and the aspartate ammonia lyase gene aspA derived from Escherichia coli. output.

Preferably, the expression vector of the recombinant gene is pRSFDuet-1.

Preferably, the specific process of replacing the growth-dependent promoter to respectively regulate the expression of the diaminopimelic acid decarboxylase gene lysA and the homoserine dehydrogenase gene metL is: replacing the diaminopimelic acid decarboxylase gene derived from Escherichia coli lysA and/or the homoserine dehydrogenase gene metL derived from Escherichia coli are integrated into the MCS I region and/or MCS II region of the pETDuet-1 expression vector, and the growth promoter is replaced by the T7 promoter in the gene integration region through homologous recombination. son.

During the experimental exploration process, the inventors found that directly knocking out the diaminopimelate decarboxylase gene lysA and the homoserine dehydrogenase gene metL in E. coli will lead to the loss of key branch pathways, resulting in amino acid deficiency to a certain extent. It will inhibit the growth of bacteria, thereby affecting the production of ectoine. Therefore, the inventors replaced the promoters of the competing pathway metabolic enzymes lysA and metL and regulated lysA and metL through growth-dependent promoters, which can make the growth status of the bacteria more reasonable, thereby effectively increasing the production of ectoine.

Preferably, the starting strain of the E. coli engineering strain is E. coli BL21 (DE3), and the genotype of the E. coli BL21 (DE3) is E. coli BL21 (DE3)ΔlysAΔmetL.

The present invention produces ectoine by directly knocking out the key metabolic enzyme genes lysA and metL of the competition branch. It is found that this approach is reasonable at the design level, but has a negative impact on bacterial growth. The products of the branch pathway are beneficial to bacterial growth, and once knocked out, it will affect bacterial growth; therefore, the present invention uses E. coli BL21 (DE3) ΔlysA ΔmetL as the starting strain, complements the missing genes of lysA and metL, and replaces the original promoters of lysA and metL for regulation to obtain a high-yield ectoine engineering strain.

In a second aspect, the present invention also provides a method for constructing the above-mentioned ectoine-producing Escherichia coli engineered bacteria, comprising the following steps:

(1) Integrate the recombinant gene into the expression vector to construct the recombinant expression vector I;

(2) Integrate the E. coli-derived diaminopimelate decarboxylase gene lysA and/or the E. coli-derived homoserine dehydrogenase gene metL into the MCS I region and/or MCS II region of the pETDuet-1 expression vector, Replace the T7 promoter in the gene integration region with the growth promoter through homologous recombination to construct a recombinant expression vector II;

(3) Transfer the above-mentioned recombinant expression vectors I and II into the E. coli starting strain in step to obtain the E. coli engineering strain.

In a third aspect, the present invention also provides the application of the above-mentioned Escherichia coli engineering bacteria in the production of ectoine.

Preferably, said production includes the following steps:

(1) Seed culture: activate the E. coli engineered bacteria, pick a single colony and inoculate it into the seed culture medium, and culture it at 35-39°C, 200-240 rpm for 5-7 hours;

(2) Fermentation culture: Insert the seed culture into the batch fermentation medium with an inoculation amount of 20%, add 1 mL of trace elements, culture at 35-39°C for 5-7 hours, add 150 ml of 0.2 mmol/L IPTG to the Induced and cultured at 35-39℃, it is ready;

The ingredients of the batch fermentation medium are: Na ₂ HPO ₄ ·12H ₂ O 17.9g/L, KH ₂ PO ₄ 3.1g/L, NH ₄ Cl2.0g/L, (NH ₄ ) ₂ HPO ₄ 1.0g/ L, trisodium citrate dihydrate 2.2g/L, yeast extract 2.0g/L, glycerin 30g/L, tryptone 15g/L, adjust the pH to 7.0;

The trace element components are: ferric ammonium citrate 5.6g/L, zinc sulfate heptahydrate 0.9g/L, CoCl ₂ ·6H ₂ O 0.2g/L, manganese chloride tetrahydrate 1.0g/L, CuCl ₂ ·2H ₂ O 0.10g/L, boric acid 0.2g/L, Na ₂ MoO ₄ ·2H ₂ O0.2g/L.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention uses Escherichia coli BL21 (DE3) as the starting strain to overexpress gene cluster ectABC, aspartate kinase gene lysC and aspartate ammonia lyase gene aspA, and for the first time uses growth-dependent promoters fliA, fliC, and flgC to respectively regulate the expression of diaminopimelate decarboxylase gene lysA and homoserine dehydrogenase gene metL. This combination of regulation achieves heterologous expression of ectoine without affecting the production of key amino acids to meet the bacterial requirements. While growing, the yield of ectoine was effectively increased.

Description of drawings

Figure 1 is a diagram of the biosynthetic pathways related to ectoine in recombinant bacteria and the metabolic engineering strategies involved in the present invention.

Detailed ways

In order to make the purpose, technical solution and effect of the present invention clearer and clearer, the present invention will be further described in detail below with reference to the examples. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

Example 1. Construction of E. coli engineered bacteria that biosynthesize ectoine

1. Construction of pRSF-ectABC-lysC-aspA expression plasmid

(1) Integrate the Halomonas elongata-derived gene cluster ectABC into the pRSFDuet-1 plasmid and construct it through General Company. The constructed plasmid is pRSF-ectABC;

(2) Design the primer pair lysC-F/lysC-R to clone the aspartokinase gene lysC derived from Corynebacterium glutamicum, and connect the cloned lysC fragment to the first fragment of the plasmid pRSF-ectABC through homologous recombination technology Behind the T7 promoter, the constructed plasmid is pRSF-ectABC-lysC;

(3) Design a primer pair pRSF-F/pRSF-R, and use the plasmid pRSF-ectABC-lysC as a template to perform PCR amplification to obtain a linearized vector. Simultaneously, design a primer pair aspA-F/aspA-R to clone the aspartate ammonia lyase gene aspA from Escherichia coli, and connect the cloned aspA fragment with the previous linearized plasmid vector through homologous recombination technology to obtain the pRSF-ectABC-lysC-aspA plasmid.

2. Construction of pET- _PGPPs -lysA, pET- _PGPPs -metL, and pET- _PGPPs -lysA- _PGPPs -metL expression vectors

(1) Integrate the diaminopimelic acid decarboxylase gene lysA derived from E. coli into the MCS I region of the pETDuet-1 plasmid, integrate the homoserine dehydrogenase gene metL into the MCS II region of the pETDuet-1 plasmid, and lysA and metL were integrated into the MCS I region and MCS II region of the pETDuet-1 plasmid respectively, and were constructed by General Company, and the constructed plasmids were pET-lysA, pET-metL, and pET-lysA-metL respectively;

(2) Growth-dependent promoters (GPPs) include fliA, fliC, and flgC; the fliA, fliC, and flgC promoter sequences were synthesized by General Company respectively, and their nucleotide sequences are as shown in SEQ ID NO: 1-3 in the sequence listing. As shown, design primer pairs fliA-F1/fliA-R1, fliA-F2/fliA-R2, fliC-F1/fliC-R1, fliC-F2/fliC-R2, flgC-F1/flgC-R1, flgC-F2/ flgC-R2;

1. Use the primer pair fliA-F1/fliA-R1 to PCR amplify the pET-lysA plasmid to obtain a linearized vector, and connect the fliA promoter sequence to the T7 promoter in the MCS I region of the linearized plasmid through homologous recombination technology The position of the subon was used to obtain the plasmid pET-P _fliA -lysA;

2. PCR amplification of the pET-lysA plasmid was performed using the primer pair fliC-F1/fliC-R1 to obtain a linearized vector, and the fliC promoter sequence was connected to the position of the T7 promoter in the MCS I region of the linearized plasmid by homologous recombination technology to obtain the plasmid pET-P _fliC -lysA;

3. PCR amplification of the pET-lysA plasmid was performed using primer pair flgC-F1/flgC-R1 to obtain a linearized vector, and the flgC promoter sequence was connected to the position of the T7 promoter in the MCS I region of the linearized plasmid by homologous recombination technology to obtain the plasmid pET-P _flgC -lysA;

4. Use the primer pair fliA-F2/fliA-R2 to PCR amplify the pET-metL plasmid to obtain a linearized vector, and connect the fliA promoter sequence to the T7 promoter in the MCS II region of the linearized plasmid through homologous recombination technology The position of the subon was used to obtain the plasmid pET-P _fliA -metL;

5. Use the primer pair fliC-F2/fliC-R2 to PCR amplify the pET-metL plasmid to obtain a linearized vector, and connect the fliC promoter sequence to the T7 promoter of the MCS II region of the linearized plasmid through homologous recombination technology The position of the subunit was used to obtain the plasmid pET-P _fliC -metL;

6. Use the primer pair flgC-F2/flgC-R2 to PCR amplify the pET-metL plasmid to obtain a linearized vector, and connect the flgC promoter sequence to the T7 promoter of the MCS II region of the linearized plasmid through homologous recombination technology The position of the subon was used to obtain the plasmid pET-P _flgC -metL;

7. Use primer pairs fliA-F1/fliA-R1 and fliA-F2/fliA-R2 to perform PCR amplification on the pET-lysA-metL plasmid in steps to obtain a linearized vector, and connect the fliA promoter sequence to the T7 promoter position of the linearized plasmid MCS I region and MCS II region respectively by homologous recombination technology to obtain the plasmid pET-P _fliA -lysA-P _fliA -metL;

8. Use the primer pair fliA-F1/fliA-R1 and fliC-F2/fliC-R2 to perform step-by-step PCR amplification of the pET-lysA-metL plasmid to obtain a linearized vector, and use homologous recombination technology to combine fliA and fliC The promoter sequence was connected to the position of the T7 promoter in the linearized plasmid MCS I region and MCS II region respectively, to obtain the plasmid pET-P _fliA -lysA-P _fliC -metL;

9. Use the primer pair fliA-F1/fliA-R1 and flgC-F2/flgC-R2 to perform step-by-step PCR amplification of the pET-lysA-metL plasmid to obtain a linearized vector, and use homologous recombination technology to combine fliA and flgC The promoter sequence was connected to the position of the T7 promoter in the linearized plasmid MCS I region and MCS II region respectively to obtain plasmid pET-P _fliA -lysA-P _flgC -metL;

10. Use the primer pair fliC-F1/fliC-R1 and fliA-F2/fliA-R2 to perform step-by-step PCR amplification of the pET-lysA-metL plasmid to obtain a linearized vector, and use homologous recombination technology to combine fliC and fliA The promoter sequence was connected to the position of the T7 promoter in the linearized plasmid MCS I region and MCS II region respectively, to obtain the plasmid pET-P _fliC -lysA-P _fliA -metL;

11. Use the primer pairs fliC-F1/fliC-R1 and fliC-F2/fliC-R2 to perform step-by-step PCR amplification of the pET-lysA-metL plasmid to obtain a linearized vector, and use homologous recombination technology to convert the fliC promoter The sequences were connected to the positions of the T7 promoter in the MCS I region and MCS II region of the linearized plasmid, respectively, to obtain the plasmid pET-P _fliC -lysA-P _fliC -metL;

12. Use the primer pair fliC-F1/fliC-R1 and flgC-F2/flgC-R2 to perform step-by-step PCR amplification of the pET-lysA-metL plasmid to obtain a linearized vector, and use homologous recombination technology to combine fliC and flgC The promoter sequence was connected to the position of the T7 promoter in the linearized plasmid MCS I region and MCS II region respectively to obtain plasmid pET-P _fliC -lysA-P _flgC -metL;

13. Use the primer pair flgC-F1/flgC-R1 and fliA-F2/fliA-R2 to perform step-by-step PCR amplification of the pET-lysA-metL plasmid to obtain a linearized vector, and use homologous recombination technology to combine flgC and fliA The promoter sequence was connected to the position of the T7 promoter in the linearized plasmid MCS I region and MCS II region respectively to obtain plasmid pET-P _flgC -lysA-P _fliA -metL;

14. The pET-lysA-metL plasmid was amplified by PCR step by step using primer pairs flgC-F1/flgC-R1 and fliC-F2/fliC-R2 to obtain a linearized vector, and the flgC and fliC promoter sequences were connected to the positions of the T7 promoter in the MCS I region and MCS II region of the linearized plasmid respectively by homologous recombination technology to obtain the plasmid pET-P _flgC -lysA-P _fliC -metL;

15. The pET-lysA-metL plasmid was amplified by PCR step by step using primer pairs flgC-F1/flgC-R1 and flgC-F2/flgC-R2 to obtain a linearized vector, and the flgC promoter sequence was connected to the position of the T7 promoter in the MCS I region and MCS II region of the linearized plasmid by homologous recombination technology to obtain the plasmid pET-P _flgC -lysA-P _flgC -metL;

3. Construction of different recombinant E. coli engineering bacteria

(1) The CRISPER method was used to knock out the diaminopimelate decarboxylase gene lysA and the homoserine dehydrogenase gene metL to obtain gene-knockout Escherichia coli E. coli BL21(DE3)ΔlysA, E. coli BL21(DE3)ΔmetL, and E. coli BL21(DE3)ΔlysAΔmetL, respectively;

(2) Mix the obtained recombinant vector pRSF-ectABC with E.coli BL21(DE3)ΔlysA, E.coliBL21(DE3)ΔmetL, and E.coli BL21(DE3)ΔlysAΔmetL competent cells respectively and place them in ice for 30 minutes. Then quickly heat shock (42°C, 90s), then quickly place it on ice and let it stand for 2 minutes. Then aseptically add 1mL of LB liquid culture medium, and resume culturing for 1 hour at 37°C, 220rpm to obtain strain ECT-01. , ECT-02, ECT-03; perform the same operation on plasmid pRSF-ectABC-lysC-aspA to obtain strains ECT-04, ECT-05, and ECT-06;

(3) pET-P _fliA -lysA, pET-P _fliC -lysA, and pET-P _flgC -lysA were respectively transformed into strain ECT-04 to obtain strains ECT-07, ECT-08, and ECT-09; pET-P _fliA -metL, pET-P _fliC -metL, and pET-P _flgC -metL were respectively transformed into strain ECT-05 to obtain strains ECT-10, ECT-11, and ECT-12; pET-P _fliA -lysA-P _fliA -metL, pET-P _fliA -lysA-P _fliC -metL, pET-P _fliA -lysA-P _flgC -metL, pET-P _fliC -lysA-P _fliA -metL, pET-P _fliC -metL, pET-P _{fliC -lysA} -P _flgC -metL, and pET-P _flgC -lysA-P _fliA -metL, pET-P _flgC -lysA-P _fliC -metL, and pET-P _flgC -lysA-P _flgC -metL were respectively transformed into the ECT-06 strain to obtain strains ECT-13, ECT-14, ECT-15, ECT-16, ECT-17, ECT-18, ECT-19, ECT-20, and ECT-21.

The primer pairs described in the examples are shown in Table 1.

Table 1

Primer name Primer sequences lysC-F CCACAGCCAGGATCCGAATTCGATGGCCCTGGTCGTACAG lysC-R GCATTATGCGGCCGCAAGCTTTTAGCGTCCGGTGCCTGC pRSF-F CTGATGAAAGCGAACAGTAACATCATCACCACAGCCAGGA pRSF-R ATACGAATGTTGTTTGACATGTGATGGCTGCTGCCCATGG aspA-F ATGTCAAACAACATTCGTAT aspA-R TTACTGTTCCGCTTTCATCAG fliA-F1 ATAACGCAGGGCTGTTTATCGGAATTGTGAGCGGATAACA fliA-R1 ATTAGTGGGTGAAATGAGGGATTTCGCGGGATCGAGATCG fliC-F1 GCGATTGAGCCGACGGGTGGGGAATTGTGAGCGGATAACA fliC-R1 TTTCAAAAACAGCCATTTTTATTTCGCGGGATCGAGATCG flgC-F1 TTGATACCTGCGGAGGAGATGGAATTGTGAGCGGATAACA flgC-R1 GAATAAACGCAAAATGGGTCATTTCGCGGGATCGAGATCG fliA-F2 ATAACGCAGGGCTGTTTATCGGAATTGTGAGCGGATAACA fliA-R2 ATTAGTGGGTGAAATGAGGGATTTCGATTATGCGGCCGTG fliC-F2 GCGATTGAGCCGACGGGTGGGGAATTGTGAGCGGATAACA fliC-R2 TTTCAAAAACAGCCATTTTTATTTCGATTATGCGGCCGTG flgC-F2 TTGATACCTGCGGAGGAGATGGAATTGTGAGCGGATAACA flgC-R2 GAATAAACGCAAAATGGGTCATTTCGATTATGCGGCCGTG

Example 2. Fermentation of Escherichia coli engineered strains to produce ectoine (ECT)

The specific methods are as follows:

(1) The composition of batch fermentation medium is: Na ₂ HPO ₄ ·12H ₂ O 17.9g/L, KH ₂ PO ₄ 3.1g/L, NH ₄ Cl2.0g/L, (NH ₄ ) ₂ HPO ₄ 1.0g /L, trisodium citrate dihydrate 2.2g/L, yeast extract 2.0g/L, glycerin 30g/L, tryptone 15g/L, adjust the pH to 7.0, and dilute to volume with deionized water.

(2) Pick a single colony of genetically engineered bacteria that produce ectoine and put it into a 250mL round-bottomed flask containing batch fermentation seed culture medium. Use the seed culture medium to fill 50mL of liquid and heat at 37°C, 220rpm/min. Cultivate for 6 hours, in which the seed medium is LB culture medium.

(3) Perform batch fermentation culture, insert the seed culture into a 1.5L shake flask containing 100mL of batch fermentation medium at an inoculation amount of 20%, add 1mL of trace elements while inoculating, and culture at 37°C for 6 hours. Add 0.2 mmol/L IPTG 150 ml to induce culture at 37°C; the trace element composition is: ferric ammonium citrate 5.6 g/L, zinc sulfate heptahydrate 0.9 g/L, CoCl ₂ ·6H ₂ O 0.2 g/L, tetrahydrate Manganese chloride 1.0g/L, CuCl ₂ ·2H ₂ O 0.10g/L, boric acid 0.2g/L, Na ₂ MoO ₄ ·2H ₂ O 0.2g/L.

(4) Centrifuge the fermentation broth, and use high-performance liquid chromatography to determine the concentration of ectoine. The ectoine production of different engineering strains is shown in Table 2.

Table 2

As can be seen from Table 2, the expression effect of plasmid pRSF-ectABC-lysC-aspA is significantly better than that of plasmid pRSF-ectABC, that is, the gene cluster ectABC, aspartokinase gene lysC and aspartate are overexpressed in E. coli engineering bacteria The acid-ammonium lyase gene aspA constructs a biological metabolic pathway for ectoine in the bacteria, achieving heterologous synthesis of ectoine and further increasing the production of ectoine; and, research has found that strains lacking both lysA and metL genes The yield in ECT-03 was obviously limited, but after strengthening the pathway construction, the yield was significantly increased (ECT-06), indicating that based on the overexpression of the gene cluster ectABC, the simultaneous expression of lysC and aspA can further increase the yield of ectoine. ; In addition, by carrying out gene backfilling in strains lacking lysA and metL genes, and using growth-dependent promoters to control gene expression, ECT production was improved. After deletion of single genes lysA or metL, respectively, the backfilling was at a certain level. The yield of ECT was improved to a certain extent, and the yield was further improved after the defects of the two genes were compensated at the same time. In the replaced growth-dependent promoters fliA, fliC, and flgC, the fliC or flgC promoters were respectively regulated to improve the four-fold improvement. The effect on hydrogen pyrimidine yield is even more significant.

In summary, the present invention uses Escherichia coli BL21 (DE3) as the starting strain to overexpress the gene cluster ectABC, aspartate kinase gene lysC and aspartate ammonia lyase gene aspA, and uses the growth-dependent promoter fliA , fliC, and flgC to respectively regulate the expression of diaminopimelate decarboxylase gene lysA and homoserine dehydrogenase gene metL. This combination of regulation achieves heterologous expression of ectoine without affecting the production of key amino acids. Thus, while satisfying the growth of bacterial cells, the production of ectoine is effectively increased.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention may be modified or equivalently substituted without departing from the essence and scope of the technical solution of the present invention.

Claims (10)

1. The escherichia coli engineering bacterium for producing the tetrahydropyrimidine is characterized in that the escherichia coli engineering bacterium is used for respectively regulating and controlling the expression of a diaminopimelate decarboxylase gene lysA and/or a homoserine dehydrogenase gene metL by using a growth-dependent promoter; the escherichia coli engineering bacteria overexpress recombinant genes, and the recombinant genes comprise gene cluster ectABC.

2. The engineered escherichia coli of claim 1, wherein the growth-dependent promoter comprises at least one of a fliA, fliC, flgC promoter; the nucleotide sequence of the fliA promoter is shown in SEQ ID NO:1, the nucleotide sequence of the fliC promoter is shown in SEQ ID NO:2, the nucleotide sequence of the flgC promoter is shown in SEQ ID NO: 3.

3. The escherichia coli engineering bacterium according to claim 2, wherein the growth-dependent promoter comprises at least one of fliC and flgC promoter.

4. The engineered escherichia coli of claim 1, wherein the recombinant genes further comprise aspartokinase gene lysC and aspartokinase gene aspA.

5. The engineered escherichia coli of claim 1, wherein the expression vector of the recombinant gene is prsduet-1.

6. The escherichia coli engineering bacterium according to claim 1, wherein the specific process of the replacement growth-dependent promoter for respectively regulating the expression of the diaminopimelate decarboxylase gene lysA and the homoserine dehydrogenase gene metL is as follows: the diaminopimelate decarboxylase gene lysA from E.coli and/or homoserine dehydrogenase gene metL from E.coli were integrated into the MCS I and/or MCS II regions of the pETDuet-1 expression vector and the growth promoter was replaced by homologous recombination with the T7 promoter of the gene integration region.

7. The escherichia coli engineering bacterium according to claim 1, wherein the initial strain of the escherichia coli engineering bacterium is escherichia coli BL21 (DE 3), and the genotype of the escherichia coli BL21 (DE 3) is E.coli BL21 (DE 3) delta lysA delta metL.

8. The method for constructing engineering bacteria of escherichia coli producing tetrahydropyrimidine according to any one of claims 1 to 7, comprising the following steps:

(1) Integrating the recombinant gene onto an expression vector to construct a recombinant expression vector I;

(2) Integrating a diaminopimelate decarboxylase gene lysA derived from escherichia coli and/or a homoserine dehydrogenase gene metL derived from escherichia coli into an MCS I region and/or an MCS II region of the pETDuet-1 expression vector, and replacing a growth promoter with a T7 promoter of the gene integration region through homologous recombination to construct a recombinant expression vector II;

(3) Transferring the recombinant expression vectors I and II into a starting strain of the escherichia coli in the step to obtain the escherichia coli engineering bacteria.

9. Use of the escherichia coli engineering bacteria of any one of claims 1-7 in the production of tetrahydropyrimidine.

10. The use according to claim 9, wherein the production comprises the steps of:

(1) Seed culture: activating the escherichia coli engineering bacteria, picking single bacterial colonies, inoculating the single bacterial colonies into a seed culture medium, and culturing for 5-7h at 35-39 ℃ and 200-240 rpm;

(2) Fermentation culture: inoculating the seed culture into batch fermentation medium with 20% inoculum size, adding 1mL trace element, culturing at 35-39deg.C for 5-7 hr, adding 0.2mmol/L IPTG 150mL, and inducing culturing at 35-39deg.C;

the batch fermentation medium comprises the following components: na (Na) ₂ HPO ₄ ·12H ₂ O 17.9g/L、KH ₂ PO ₄ 3.1g/L，NH ₄ Cl2.0g/L、(NH ₄ ) ₂ HPO ₄ 1.0g/L, trisodium citrate dihydrate 2.2g/L, yeast extract 2.0g/L,30g/L glycerol and 15g/L tryptone, and adjusting the pH value to 7.0;

the trace elements comprise the following components: ferric ammonium citrate 5.6g/L, zinc sulfate heptahydrate 0.9g/L, coCl ₂ ·6H ₂ O0.2g/L, manganese chloride tetrahydrate 1.0g/L, cuCl ₂ ·2H ₂ O0.10 g/L, boric acid 0.2g/L, na ₂ MoO ₄ ·2H ₂ O0.2g/L。