Improved promoter and method for producing lysine using the same

Abstract

Improved promoter and method for producing lysine using the same. The present invention proposes an improved strategy for promoter construction, namely selecting a partial sequence at the 5' end of the promoter native gene as a "buffer zone" between the promoter and the downstream target gene. Compared with the traditional promoter construction method, the improved method has better effect, and the same promoter shows better stability when expressing different genes. In addition to providing an improved promoter construction strategy, the present invention also provides an improved promoter, and the use of the improved promoter in overexpressing the key genes lysC-asd, dapA and ddh in the lysine synthesis pathway, and the use of the improved promoter in the production of lysine.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an improved promoter and a method for producing lysine using the same. Background Art

[0002] Lysine is one of the essential amino acids for humans and mammals. It cannot be synthesized in the body and must be supplemented from food. Lysine has the functions of promoting human growth and development, enhancing immunity, resisting viruses, promoting fat oxidation, relieving anxiety, etc., and has a positive effect on human and animal health. Because of its essential amino acid characteristics and functionality, lysine has become the world's second largest amino acid production variety and is widely used in animal feed, medicine and food industries. About 90% of it is used in the feed industry and 10% in the food and pharmaceutical industries. As an animal feed additive, lysine can help the body absorb other amino acids, thereby improving feed quality.

[0003] Because lysine needs to be produced on a large scale, lysine is mainly produced by microbial fermentation, which has the advantages of low raw material cost, mild reaction conditions, and easy large-scale production. However, the fermentation performance and conversion rate of lysine strains are still relatively large. By enhancing the expression of some genes in the amino acid metabolic pathway, the yield and conversion rate of lysine can be improved. The enhanced gene expression in some key positions can improve the expression level of the enzyme and the anti-feedback ability of gene transcription. Patent CN202311376891.6 discloses a promoter used in Corynebacterium glutamicum, which can enhance the expression of the lysC gene, thereby enhancing the lysine production capacity of the production strain.

[0004] However, most promoters are specific and have different expressions in different target genes, so a promoter with better applicability is needed.

[0005] In this context, the present invention constructs an improved promoter based on the promoter of the pgk gene in Corynebacterium glutamicum. It is found that the promoter has a good expression effect in different target genes and has a significant effect on increasing the production of lysine. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides an improved strategy for promoter construction, an improved promoter, and the use of the improved promoter in overexpressing key genes lysC-asd, dapA and ddh in the lysine biosynthesis pathway, as well as the use of the improved promoter in producing lysine.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides an improved promoter construction strategy. We designed Figure 2 The improved promoter construction strategy shown is to cut off part of the 5' end sequence of the native gene expressed by the candidate promoter (the length of the cut sequence is a multiple of 3, that is, it contains a complete triplet code, and the length is about 100bp) and form an improved promoter together with the candidate promoter, and add a stop codon (TAA, TAG, TGA) and a ribosome binding site (RBS) for translation of the reporter gene or the target gene downstream of the cut gene.

[0009] Preferably, the improved promoter construction strategy is to additionally intercept a sequence of 80-500 bp in length at the 5' end of the pgk gene on the basis of the 216 bp upstream of the ATG of the pgk gene of Corynebacterium glutamicum, preferably a sequence of 90-300 bp in length, and more preferably a sequence of 117 bp in length;

[0010] Preferably, a stop codon and / or a ribosome binding site is further added downstream of the 5' end partial sequence of the native gene;

[0011] Preferably, the stop codon is TAA, TAG or TGA.

[0012] More preferably, a TAG stop codon and a ribosome binding site having the sequence 5'-AACGAAAGGAAACACTCAT-3' are added downstream of the above-mentioned truncated sequence.

[0013] In a second aspect, the present invention provides an improved promoter, denoted as promoter Ppgk100, whose nucleotide sequence is shown in SEQ ID NO:2.

[0014] The improved promoter Ppgk100 is obtained according to the improved promoter construction strategy described in the first aspect above.

[0015] In a third aspect, the present invention provides an expression vector comprising the improved promoter described in the second aspect.

[0016] In a fourth aspect, the present invention provides a host cell comprising the improved promoter described in the second aspect or the expression vector described in the third aspect;

[0017] Preferably, the host cell is a microorganism of the genus Corynebacterium, more preferably, the host cell is Corynebacterium glutamicum.

[0018] In a fifth aspect, the present invention provides use of the improved promoter Ppgk100 described in the second aspect, the expression vector described in the third aspect, or the host cell described in the fourth aspect in producing lysine.

[0019] In a sixth aspect, the present invention provides the use of the improved promoter Ppgk100 as described in the second aspect, or the expression vector as described in the third aspect, or the host cell as described in the fourth aspect in overexpressing key genes in the lysine synthesis pathway;

[0020] Preferably, the key gene in the lysine synthesis pathway is lysC-asd;

[0021] Preferably, the key gene in the lysine biosynthesis pathway is dapA;

[0022] Preferably, the key gene in the lysine biosynthesis pathway is ddh.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention aims at the problem of unstable promoter expression level that often occurs in promoter screening and application, and proposes an improved strategy for promoter construction, that is, selecting a partial sequence at the 5' end of the promoter native gene as a "buffer zone" between the promoter and the downstream target gene. The improved promoter shows more excellent and stable performance and higher amino acid yield in overexpressing genes. The improved promoter Ppgk100 is used to overexpress lysC-asd, dapA and ddh genes, which can significantly increase the lysine yield, and the overexpression performance of the Ppgk100 promoter for different genes is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the traditional promoter screening and application process;

[0026] Figure 2 It is a comparison chart of traditional promoter screening and improved promoter screening;

[0027] Figure 3 The egfp reporter gene was used to verify the expression level of the Ppgk and Ppgk100 promoters;

[0028] Figure 4 This is a schematic diagram of using the improved promoter to overexpress the lysC-asd gene. DETAILED DESCRIPTION

[0029] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0030] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0031] Example 1 Improved promoter screening method and improved Corynebacterium glutamicum Ppgk100 promoter

[0032] (I) Design of control group promoter sequence and improved promoter sequence.

[0033] We design Figure 2 The improved promoter screening method shown is to cut off a partial sequence at the 5' end of the native gene expressed by the candidate promoter (the length of the cut sequence is a multiple of 3, i.e., it contains a complete triplet code and is about 100 bp in length) and combine it with the candidate promoter to form an improved promoter, and add a stop codon (TAA, TAG, TGA) and a ribosome binding site (RBS) for translation of the reporter gene or the target gene downstream of the cut gene.

[0034] Taking the strong promoter pgk gene promoter Ppgk in Corynebacterium glutamicum as an example, the control group (i.e., the traditional strategy) uses the 216bp upstream of the ATG of the pgk gene as the promoter region, denoted as promoter Ppgk, and its nucleotide sequence is shown in SEQ ID NO: 1. The improved promoter construction strategy not only intercepts the 117bp sequence at the 5' end of the pgk gene in addition to the 216bp Ppgk promoter sequence, but also adds a TAG stop codon and a ribosome binding site (RBS) with a sequence of 5'-AACGAAAGGAAACACTCAT-3' downstream, together forming a new "promoter", denoted as promoter Ppgk100, and its nucleotide sequence is shown in SEQ ID NO: 2.

[0035] (II) Construction of promoter screening and characterization plasmids pEC-XK99E-Ppgk-egfp and pEC-XK99E-Ppgk100-egfp. Using pEC-XK99E plasmid as template and SEQ ID NO:3 and SEQ ID NO:4 as primers, PCR amplification of vector pEC-XK99E was performed; using the genome of Corynebacterium glutamicum ATCC13032 as template and SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:5 and SEQ ID NO:7 as primers, Ppgk and Ppgk100 promoters were amplified by PCR, where Ppgk and Ppgk100 promoters have homology arms recombined with egfp; using pEGFP-C1 as template and SEQ ID NO:8 and SEQ ID NO:9 as primers, PCR amplification of egfp gene was performed, and the four PCR products were recovered by gel. Then, 2X MultiF Seamless Assembly Mix (ABclonal, Cat. No.: RK21020) was used to assemble three fragments of DNA, namely, the three-fragment assembly of pEC-XK99E vector + Ppgk + egfp and the three-fragment assembly of pEC-XK99E vector + Ppgk100 + egfp. The assembled reaction solution was then transformed into E. coli DH5α to construct promoter screening and characterization plasmids pEC-XK99E-Ppgk-egfp and pEC-XK99E-Ppgk100-egfp.

[0036] Table 1

[0037]

[0038] The underlined sequence is the 40 bp homology arm required for DNA assembly, the italicized sequence is the artificially added RBS region, and the lowercase letter is the stop codon TAG.

[0039] (III) Results. The successfully constructed and correctly sequenced plasmid was electroporated into Corynebacterium glutamicum ATCC13032, and the fluorescence value of the strain was then measured by a fluorescence spectrophotometer to reflect the transcription level of the promoter. Figure 3 As shown, the improved promoter has an EGFP fluorescence value that is 37.4% higher than that of the traditional model, indicating that the improved promoter screening method proposed in the present invention is feasible and that the reporter gene expression level obtained by the improved method is significantly improved.

[0040] Example 2 Overexpression of key genes lysC and asd in the lysine biosynthesis pathway using the improved promoter

[0041] The lysC gene encoding aspartate kinase and the asd gene encoding aspartate semialdehyde dehydrogenase exist in the form of a tandem operon on the genome of Corynebacterium glutamicum ATCC13032, and can be controlled by a promoter. In order to detect and verify the stability of the improved promoter, the lysine yield was detected by overexpression of the lysC-asd gene to determine the performance stability of the promoter constructed in Example 1 when expressing different genes.

[0042] (I) Lysine overexpression plasmids pEC-XK99E-Ppgk-lysC-asd and pEC-XK99E-Ppgk100-lysC-asd were constructed. The pEC-XK99E plasmid was used as a template and SEQ ID NO:3 and SEQ ID NO:4 were used as primers to PCR amplify the vector pEC-XK99E; the genome of Corynebacterium glutamicum ATCC13032 was used as a template and SEQ ID NO:5 and SEQ ID NO:10, SEQ ID NO:5 and SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 were used as primers to PCR amplify the Ppgk, Ppgk100 promoter, and lysC-asd fragment, wherein the Ppgk and Ppgk100 promoters have homology arms recombined with lysC.

[0043] The PCR products were recovered by gel recovery, and then three-fragment DNA assembly was performed using 2X MultiF Seamless Assembly Mix (ABclonal, Cat. No.: RK21020), i.e., three-fragment assembly of pEC-XK99E vector + Ppgk + lysC-asd and three-fragment assembly of pEC-XK99E vector + Ppgk100 + lysC-asd. The assembled reaction solution was then transformed into E. coli DH5α to construct lysine overexpression plasmids pEC-XK99E-Ppgk-lysC-asd and pEC-XK99E-Ppgk100-lysC-asd.

[0044] Table 2

[0045]

[0046] The underlined sequence is the 40 bp homology arm required for DNA assembly, the italicized sequence is the artificially added RBS region, and the lowercase letter is the stop codon TAG.

[0047] (II) The successfully constructed and correctly sequenced plasmid was electrotransformed into Corynebacterium glutamicum ATCC13032, and then fermented to detect lysine production. Wild strains of Corynebacterium glutamicum ATCC13032, ATCC13032-Ppgk-lysC-asd and ATCC13032-Ppgk100-lysC-asd strains introduced with overexpression plasmids were inoculated into 250 mL Erlenmeyer flasks containing 30 ml of seed culture medium, and kanamycin antibiotics were added to the seed culture medium to maintain the stable presence of the overexpression plasmid in the strain. No antibiotics were added to the wild strain. After 8 hours of seed culture, the fermentation medium was transferred for fermentation.

[0048] Seed culture medium (pH 7.3): 20g sucrose, 10g peptone, 5g yeast extract powder, 5g ammonium sulfate, 3.5g urea, 0.5g cysteine, 10.5g potassium dihydrogen phosphate, 120mg ferrous sulfate, 120mg manganese sulfate, 6mg zinc sulfate, 6mg copper sulfate, 4.5mg VB1, 9mg calcium pantothenate, 60mg nicotinamide, 1.8mg biotin (dissolved in 1L distilled water)

[0049] Fermentation medium: (pH 7.0) glucose 70g, ammonium sulfate 40g, soy protein 2.5g, corn steep liquor 5g, urea 3g, potassium dihydrogen phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 0.1mg, VB1 1mg, calcium pantothenate 1mg, nicotinamide 3mg, calcium carbonate 30g (dissolved in 1L distilled water, adjust pH first, then add calcium carbonate)

[0050] (III) Results: After 46 h of fermentation, the lysine production was measured using a biosensor. The results are shown in Table 3.

[0051] Table 3

[0052]

[0053] The fermentation results showed that the wild-type ATCC13032 strain produced almost no lysine. The traditional promoter was used to overexpress the lysC-asd gene to obtain a lysine yield of 3.0 g / L, while the improved promoter was used to overexpress the lysC-asd gene to obtain a lysine yield of 5.5 g / L, an increase of 83.3% in lysine yield. This showed that the improved promoter construction strategy was more effective than the traditional promoter construction strategy, the Ppgk100 promoter could increase lysine yield, the performance of overexpressing lysC-asd was improved, and the overexpression performance of the Ppgk100 promoter for different genes was stable.

[0054] Example 3 Overexpression of dapA, a key gene in the lysine biosynthesis pathway, using the improved promoter

[0055] The dapA gene encodes dihydropicolinate synthase, which is responsible for catalyzing the synthesis of dihydropicolinate from aspartate semialdehyde. The gene is also a key gene in the lysine synthesis pathway. The lysine production was detected by overexpression of the dapA gene to determine the performance stability of the promoter constructed in Example 1 when expressing different genes.

[0056] (I) Lysine overexpression plasmids pEC-XK99E-Ppgk-dapA and pEC-XK99E-Ppgk100-dapA were constructed. The pEC-XK99E plasmid was used as a template and SEQ ID NO:3 and SEQ ID NO:4 were used as primers to PCR amplify the vector pEC-XK99E; the genome of Corynebacterium glutamicum ATCC13032 was used as a template and SEQ ID NO:5 and SEQ ID NO:14, SEQ ID NO:5 and SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17 were used as primers to PCR amplify the Ppgk, Ppgk100 promoter, and dapA gene, wherein the Ppgk and Ppgk100 promoters have homology arms recombined with dapA.

[0057] The PCR products were recovered by gel extraction, and then three-fragment DNA assembly was performed using 2X MultiF Seamless Assembly Mix (ABclonal, Cat. No.: RK21020), i.e., three-fragment assembly of pEC-XK99E vector + Ppgk + dapA and three-fragment assembly of pEC-XK99E vector + Ppgk100 + dapA. The assembled reaction solution was then transformed into E. coli DH5α to construct lysine overexpression plasmids pEC-XK99E-Ppgk-dapA and pEC-XK99E-Ppgk100-dapA.

[0058] Table 4

[0059] The underlined sequence is the 40 bp homology arm required for DNA assembly, the italicized sequence is the artificially added RBS region, and the lowercase letter is the stop codon TAG.

[0060] (II) The successfully constructed and correctly sequenced plasmid was electrotransformed into Corynebacterium glutamicum ATCC13032, and then fermented to detect lysine production. Wild strains of Corynebacterium glutamicum ATCC13032, ATCC13032-Ppgk-dapA and ATCC13032-Ppgk100-dapA strains introduced with overexpression plasmids were inoculated into 250 mL Erlenmeyer flasks containing 30 ml seed culture medium, and kanamycin antibiotics were added to the seed culture medium to maintain the stable presence of the overexpression plasmid in the strain. No antibiotics were added to the wild strain. After 8 hours of seed culture, the fermentation medium was transferred for fermentation.

[0061] Seed culture medium (pH 7.3): 20g sucrose, 10g peptone, 5g yeast extract powder, 5g ammonium sulfate, 3.5g urea, 0.5g cysteine, 10.5g potassium dihydrogen phosphate, 120mg ferrous sulfate, 120mg manganese sulfate, 6mg zinc sulfate, 6mg copper sulfate, 4.5mg VB1, 9mg calcium pantothenate, 60mg nicotinamide, 1.8mg biotin (dissolved in 1L distilled water)

[0062] Fermentation medium: (pH 7.0) glucose 70g, ammonium sulfate 40g, soy protein 2.5g, corn steep liquor 5g, urea 3g, potassium dihydrogen phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 0.1mg, VB1 1mg, calcium pantothenate 1mg, nicotinamide 3mg, calcium carbonate 30g (dissolved in 1L distilled water, adjust pH first, then add calcium carbonate)

[0063] (III) Results: After 46 h of fermentation, the lysine production was measured using a biosensor. The results are shown in Table 5.

[0064] Table 5

[0065]

[0066] The fermentation results showed that the overexpression of dapA gene using the traditional promoter achieved a lysine yield of 1.6 g / L, while the overexpression of dapA gene using the improved promoter achieved a lysine yield of 2.2 g / L, an increase of 37.5% in lysine yield. This indicated that the improved promoter construction strategy was more effective than the traditional promoter construction strategy, that the Ppgk100 promoter could increase lysine yield, and that the performance of overexpressing dapA was improved. The overexpression performance of the Ppgk100 promoter for different genes was stable.

[0067] Example 4 Overexpression of the key gene ddh in the lysine biosynthesis pathway using the improved promoter

[0068] The ddh gene encodes diaminopimelate dehydrogenase, which is responsible for catalyzing the synthesis of racemic diaminopimelate from dihydropyridine carboxylic acid, and is also a key gene in the lysine synthesis pathway. The lysine production was detected by overexpression of the ddh gene to judge the performance stability of the promoter expression of different genes constructed in Example 1.

[0069] (I) Construction of lysine overexpression plasmids pEC-XK99E-Ppgk-ddh and pEC-XK99E-Ppgk100-ddh. Using pEC-XK99E plasmid as template and SEQ ID NO:3 and SEQ ID NO:4 as primers, PCR amplification of vector pEC-XK99E was performed; using the genome of Corynebacterium glutamicum ATCC13032 as template and SEQ ID NO:5 and SEQ ID NO:18, SEQ ID NO:5 and SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21 as primers, PCR amplification of Ppgk, Ppgk100 promoter, ddh gene, wherein Ppgk and Ppgk100 promoters have homology arms recombined with ddh.

[0070] The PCR products were recovered by gel recovery, and then three-fragment DNA assembly was performed using 2X MultiF Seamless Assembly Mix (ABclonal, Cat. No.: RK21020), i.e., three-fragment assembly of pEC-XK99E vector + Ppgk + ddh and three-fragment assembly of pEC-XK99E vector + Ppgk100 + ddh. The assembled reaction solution was then transformed into E. coli DH5α to construct lysine overexpression plasmids pEC-XK99E-Ppgk-ddh and pEC-XK99E-Ppgk100-ddh.

[0071] Table 6

[0072]

[0073] The underlined sequence is the 40 bp homology arm required for DNA assembly, the italicized sequence is the artificially added RBS region, and the lowercase letter is the stop codon TAG.

[0074] (II) The successfully constructed and correctly sequenced plasmid was electrotransformed into Corynebacterium glutamicum ATCC13032, and then fermented to detect lysine production. Wild strains of Corynebacterium glutamicum ATCC13032, ATCC13032-Ppgk-ddh and ATCC13032-Ppgk100-ddh strains introduced with overexpression plasmids were inoculated into 250 mL Erlenmeyer flasks containing 30 ml seed culture medium, and kanamycin antibiotics were added to the seed culture medium to maintain the stable presence of the overexpression plasmid in the strain. No antibiotics were added to the wild strain. After 8 hours of seed culture, the fermentation medium was transferred for fermentation.

[0075] Seed culture medium (pH 7.3): 20g sucrose, 10g peptone, 5g yeast extract powder, 5g ammonium sulfate, 3.5g urea, 0.5g cysteine, 10.5g potassium dihydrogen phosphate, 120mg ferrous sulfate, 120mg manganese sulfate, 6mg zinc sulfate, 6mg copper sulfate, 4.5mg VB1, 9mg calcium pantothenate, 60mg nicotinamide, 1.8mg biotin (dissolved in 1L distilled water)

[0076] Fermentation medium: (pH 7.0) glucose 70g, ammonium sulfate 40g, soy protein 2.5g, corn steep liquor 5g, urea 3g, potassium dihydrogen phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 0.1mg, VB1 1mg, calcium pantothenate 1mg, nicotinamide 3mg, calcium carbonate 30g (dissolved in 1L distilled water, adjust pH first, then add calcium carbonate)

[0077] (III) Results: After 46 h of fermentation, the lysine production was measured using a biosensor. The results are shown in Table 7.

[0078] Table 7

[0079]

[0080] The fermentation results showed that overexpression of the ddh gene using the traditional promoter resulted in a lysine yield of 0.9 g / L, while overexpression of the ddh gene using the improved promoter resulted in a lysine yield of 1.2 g / L, an increase of 33.3% in lysine yield. This indicated that the improved promoter construction strategy was more effective than the traditional promoter construction strategy, that the Ppgk100 promoter could increase lysine yield, and that the performance of overexpression of ddh was improved. The overexpression performance of the Ppgk100 promoter for different genes was stable.

[0081] The present invention aims at the problem of unstable promoter expression level that often occurs in promoter screening and application, and proposes an improved strategy for promoter construction, that is, selecting a partial sequence at the 5' end of the promoter native gene as a "buffer zone" between the promoter and the downstream target gene. Compared with the traditional promoter construction method, the improved method has better effect, and the same promoter shows better stability when expressing different genes. In addition to providing an improved promoter construction strategy, the present invention also provides an improved promoter, Ppgk100 promoter, and the application of the improved promoter in overexpressing the key genes lysC-asd, dapA and ddh in the lysine synthesis pathway; and the application of the improved promoter in the production of lysine.

[0082] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An improved promoter Ppgk100, characterized in that The nucleotide sequence of the promoter Ppgk100 is shown in SEQ ID NO:

2.

2. An expression vector, characterized in that: The expression vector contains the improved promoter Ppgk100 according to claim 1.

3. A host cell, characterized in that The host cell contains the improved promoter Ppgk100 according to claim 1 or the expression vector according to claim 2.

4. The host cell according to claim 3, characterized in that The host cell is Corynebacterium glutamicum.

5. Use of the improved promoter Ppgk100 according to claim 1, the expression vector according to claim 2, or the host cell according to claim 3 or 4 in producing lysine.

6. Use of the improved promoter Ppgk100 according to claim 1 or the expression vector according to claim 2 or the host cell according to claim 3 or 4 in overexpressing a key gene in a lysine synthesis pathway, wherein the key gene in the lysine synthesis pathway is lysC-asd, dapA or ddh.

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