A promoter for improving the expression level of glycinamide nucleotide synthetase and its application

Abstract

The present invention discloses a promoter for improving the expression level of glycine nucleotide synthetase and its application, belonging to the field of synthetic biology. The present invention uses the original promoter PpurD of the glycine nucleotide synthetase gene in Corynebacterium stationis ATCC 6872 as a template to construct a promoter library, uses sfGFP as a reporter protein, and adds a 180bp sequence at the 5' end of the glycine nucleotide synthetase gene through a GGGGS linker before sfGFP, and screens out a promoter PpurDmut that significantly increases the expression of the glycine nucleotide synthetase gene, and its nucleotide sequence is shown in SEQ ID NO: 1. Compared with the original promoter PpurD and the known strong promoter Psod, the promoter PpurDmut provided by the present invention can strengthen the transcription level of glycine nucleotide synthetase, thereby promoting the expression of glycine nucleotide synthetase in Corynebacterium stationis, and then can improve the metabolic flux of nucleotide products. In addition, the promoter can also improve the expression of heterologous genes in Corynebacterium stationis, and can be used as a universal promoter, with strong industrial applicability.

Description

Technical Field

[0001] The present disclosure belongs to the field of synthetic biology and relates to a promoter and an application thereof, and specifically to a promoter for increasing the transcription level of a glycinamide nucleotide synthetase gene in Corynebacterium stagnantum and an application thereof. Background Art

[0002] Nucleotides are precursors for the synthesis of DNA and RNA, and play a vital role in the composition of cells, the production and consumption of energy, metabolism, and functional regulation. Due to the many unique and important properties and functions of nucleotides, nucleotides have a wide range of applications and are widely used in food, agriculture, medicine, animal feed, and fine chemicals. The fermentation production of nucleotides by microbial strains uses simple substances such as ribose phosphate, amino acids, and one-carbon units as raw materials to synthesize nucleotides, i.e., the de novo synthesis of nucleotides, which includes the synthesis of IMP (inosine monophosphate); the generation of AMP (adenine nucleotide) and GMP (guanine nucleotide) from IMP; and the phosphorylation of nucleoside monophosphate to generate nucleoside diphosphate and nucleoside triphosphate. See also Figure 1 , Figure 1 The synthesis pathway of IMP in the de novo nucleotide synthesis pathway includes 11 steps: (1) activation of 5-phosphoribose; (2) obtaining the N9 atom of purine; (3) obtaining the C4, C5 and N7 atoms of purine; (4) obtaining the C8 atom of purine; (5) obtaining the N3 atom of purine; (6) formation of the purine imidazole ring; (7) obtaining the C6 atom of purine; (8) obtaining the N1 atom; (9) removing fumaric acid; (10) obtaining C2; (11) cyclization to generate IMP. The synthesis pathway involves the participation of multiple key enzymes. Glycineamide ribonucleotide synthetase (EC 6.3.4.13, PurD) is one of the key enzymes, and its expression level can be increased to increase the titer of the nucleotide end product.

[0003] With the development of metabolic engineering and synthetic biology, various corresponding methods have been developed for high-titer production of target products by microorganisms. Among them, promoter engineering has become a key technology for regulating gene expression and optimizing the biosynthetic pathway of target products, and is widely used in metabolic engineering transformation of microorganisms. Promoters are important elements for regulating gene expression. They are located in the non-coding region upstream of the 5' end of structural genes and are DNA fragments that are specifically recognized and bound by RNA polymerase. The activity of promoters is affected by many factors, and in heterologous expression of genes, it is often necessary to select promoters with different activities according to actual needs.

[0004] Corynebacterium microorganisms are an important genus of bacteria. Many Corynebacterium are used as industrial production model strains to achieve efficient production of multiple proteins, metabolites, etc. Among them, Corynebacterium stagnans is the main microbial strain for industrial fermentation production of nucleotide products. In Corynebacterium, many endogenous or heterologous promoters have been developed for constituting or inducing gene expression, but the strong constitutive promoters available for selection are still very limited. Among them, Psod and Peftu are the most commonly used promoters in strain development, but their promoter effects on target genes are affected by the sequence at the 5' end of the coding region and the different target genes connected thereto. When they are applied to express endogenous glycinamide nucleotide synthetase in Corynebacterium stagnans, they have problems such as low expression efficiency, resulting in a low titer of nucleotide end products in the de novo synthesis pathway, so it is still necessary to develop a strong promoter compatible with the target gene. Summary of the invention

[0005] Based on the technical problems existing in the prior art, the present invention provides a promoter with a nucleotide sequence as shown in SEQ ID NO: 1, and the promoter is operably connected to the glycinamide nucleotide synthetase gene to achieve efficient expression of the glycinamide nucleotide synthetase gene in Corynebacterium stagnantum, thereby increasing the titer of the final nucleotide final product.

[0006] The present invention provides a promoter, wherein the promoter is selected from any one of the following groups consisting of (i)-(iii):

[0007] (i) a polynucleotide having a nucleotide sequence of SEQ ID NO: 1;

[0008] (ii) a polynucleotide having a nucleotide sequence that is at least 80%, optionally at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identical to the nucleotide sequence of SEQ ID NO: 1, and having similar promoter activity;

[0009] (iii) a nucleotide sequence complementary to the nucleotide sequence shown in (i) or (ii).

[0010] The promoter provided by the present invention is obtained by mutating the nucleotides at positions 40, 42, 44 to 51 and 53 to 56 of the original promoter shown in SEQ ID NO: 2 from C, G, CGAGCGAA and AAGT to A, A, GCCCAATT and TCTG. The promoter of the present invention is screened for the adaptation of the glycineamide nucleotide synthetase gene. Compared with the original promoter or the known strong promoter Psod, the promoter of the present invention can enhance the transcription level of glycineamide nucleotide synthetase, thereby promoting the expression of glycineamide nucleotide synthetase in Corynebacterium stagnation, thereby increasing the metabolic flux of nucleotide products. In addition, the promoter of the present invention can increase the expression of heterologous genes (such as α-amylase amyE gene from Bacillus subtilis subsp.subtilis str.168) in Corynebacterium stagnation, achieving an α-amylase expression level that is 2.30 times higher than the original promoter and 2.24 times higher than the existing Corynebacterium promoter, respectively. Therefore, the promoter provided by the present invention can be used as a universal promoter and has strong industrial applicability.

[0011] Furthermore, the present invention provides a gene expression cassette, comprising the promoter and a target gene operably linked to the promoter.

[0012] Furthermore, the present invention provides a recombinant vector comprising the promoter or the gene expression cassette.

[0013] Furthermore, the present invention provides a recombinant strain comprising the gene expression cassette or the recombinant vector.

[0014] Furthermore, the recombinant strain is Corynebacterium stagnans.

[0015] Furthermore, the stagnant bacterium is stagnant bacterium ATCC 6872 or a derivative strain thereof.

[0016] Furthermore, the present disclosure provides an engineered bacterium of Corynebacterium stasis, wherein the method for constructing the engineered bacterium of Corynebacterium stasis comprises the following steps:

[0017] By gene editing technology, a promoter located upstream of the glycinamide nucleotide synthetase gene in the genome of Corynebacterium stagnantum, whose nucleotide sequence is shown in SEQ ID NO: 2, i.e., 68 bases upstream of the ATG of the glycinamide nucleotide synthetase gene, is replaced with the promoter, and positive clones are screened;

[0018] Alternatively, a gene expression cassette containing the target gene, glutamine aminotransferase, is integrated into the genome of Corynebacterium stagnans through gene editing technology, and positive clones are screened;

[0019] Alternatively, a plasmid containing a gene expression cassette in which the target gene is a transglutaminase gene is transformed into a competent bacterium of Corynebacterium stagnans, and positive clones are screened.

[0020] Furthermore, the present invention provides the use of the promoter in promoting efficient expression of a target gene in Corynebacterium stagnans.

[0021] Furthermore, the gene expression cassette, or the recombinant vector, or the recombinant strain is used in preparing proteins.

[0022] Furthermore, the protein includes glycinamide nucleotide synthetase.

[0023] Furthermore, the present invention provides a method for producing nucleotide products, which comprises culturing a recombinant strain containing the promoter operably linked to a glycinamide nucleotide synthetase gene, and collecting the produced nucleotides.

[0024] Instruction Manual

[0025] Figure 1 It is the first stage of the de novo nucleotide synthesis pathway: the synthesis of IMP (inosine monophosphate).

[0026] Figure 2 (A) is the sequence of the original promoter PpurD; (B) is the design and construction strategy of the PpurD promoter library.

[0027] Figure 3 This is a comparison chart of the fluorescence intensity of sfGFP promoted by the promoter mutant prepared in Example 1, the original promoter and the known strong promoter.

[0028] Figure 4 This is a comparison chart of the transcription levels of the glycinamide nucleotide synthetase gene in Corynebacterium stagnantum ATCC 6872 / pEC-PpurD-purD, Corynebacterium stagnantum ATCC 6872 / pEC-Psod-purD and Corynebacterium stagnantum ATCC 6872 / pEC-PpurDmut-purD in Example 2. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] The term "promoter" in the present invention refers to a nucleic acid molecule, which is usually located upstream of the target gene coding sequence, provides a recognition site for RNA polymerase, and is located upstream of the 5' direction of the mRNA transcription start site. It is a nucleic acid sequence that is not translated, and RNA polymerase binds to this nucleic acid sequence to initiate the transcription of the target gene. In the synthesis of ribonucleic acid (RNA), the promoter can interact with the transcription factors that regulate gene transcription, control the start time and degree of gene expression (transcription), and contain the core promoter region and the regulatory region, just like a "switch", which determines the activity of the gene and then controls which protein the cell starts to produce. In the sequence of the promoter, the -35 region is the recognition site of RNA polymerase, and the -10 region is the binding site of RNA polymerase.

[0031] The promoter sequence of the present invention can be modified by conventionally known mutagenesis. Therefore, the promoter may include, but is not limited to, any nucleotide sequence having 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more homology with the nucleotide sequence of SEQ ID NO: 1, and having similar promoter activity, wherein any nucleotide sequence having the above homology in which a portion of the sequence is deleted, modified, substituted or inserted should be understood to be included in the scope of the nucleic acid molecule disclosed herein, as long as the sequence has promoter activity. For example, a meaningless sequence is added to the interior or end of the corresponding nucleotide sequence of SEQ ID NO: 1, or a polynucleotide in which a portion of the sequence is deleted from the interior or end of the nucleotide sequence of SEQ ID NO: 1 is obviously also included in the scope of the present disclosure, as long as it has the same or corresponding activity as the polynucleotide.

[0032] The promoter SEQ ID NO: 2 is referred to herein as the initial promoter or original promoter, which is in contrast to the mutant promoter SEQ ID NO: 1. For the sake of convenience, the initial promoter SEQ ID NO: 2 and its mutants such as SEQ ID NO: 1 may be collectively referred to herein as "promoters".

[0033] The term "gene expression cassette" in the present invention refers to a sequence containing a promoter, a target gene or a target gene cloning site and a terminator, and in which the gene can be transcribed and translated normally. The target gene can be a protein coding gene.

[0034] The term "vector" in the present invention refers to the vehicle required to deliver the target gene to the biological cell (recipient cell) through genetic engineering. The vector used in genetic engineering is a type of self-replicating DNA molecule in which a section of DNA is removed without affecting its replication, and can be used to replace or insert exogenous (target) DNA to bring the target DNA into the host cell. Commonly used vectors include plasmids, bacteriophages, viruses, etc.

[0035] The term "derivative strain" in the present invention refers to a strain derived by any transformation, for example by one or more hybridizations and / or by mutation and / or by genetic transformation.

[0036] The gene editing technology in the present invention can adopt the Cre / loxp system, CRISPR / Cas9 system and CRISPR / Cpf1 system, etc.

[0037] The term "transformation" in the present invention refers to the process of introducing a vector including a polynucleotide encoding a target protein into a host cell, thereby being able to express the polynucleotide encoded by the protein in the host cell. The transformation method may include any method that can introduce a nucleic acid into a cell, and the transformation can be performed by selecting an appropriate standard technique known in the art according to the host cell. For example, electroporation, etc., but not limited thereto.

[0038] All primer synthesis and sequencing in the examples of the present invention were entrusted to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.

[0039] The reagents used in the embodiments of the present invention are as follows:

[0040] LB seed medium: 1% peptone, 0.5% yeast extract, 1% NaCl.

[0041] LB solid medium: 1% peptone, 0.5% yeast extract, 1% NaCl, 50ug / mL kanamycin, 2% agarose.

[0042] Fermentation medium: 20 g / L glucose; 3 g / L urea, 2 g / L NH4Cl, 1 g / L KH2PO4, 3 g / L K2HPO4, 5 g / L asparagine, 0.04 g / L L-cysteine, 0.001 g / L MnSO4·H2O, 0.001 g / L ZnSO4·7H2O, 2×10 -4 g / L CuSO4·2H2O, 0.02g / L calcium pantothenate, 0.01g / L CaCl2, 0.3g / L MgSO4, 0.01g / LFeSO4·7H2O, 6×10 -5 g / L biotin, 0.01g / L thiamine-HCl (vitamin B1 hydrochloride).

[0043] Plasmid pEC-XK99E: The public can entrust commercial companies to synthesize it according to the sequence of NCBI GenBank No. AY219683.1 (update: 05-SEP-2003).

[0044] pSenPutsfGFP: A plasmid containing the superfolded green fluorescent protein sfGFP gene preserved in the laboratory.

[0045] The GenBank accession number of the Corynebacterium stationis ATCC 6872 genome is GCA_001561975.1.

[0046] The GenBank number of glycineamide ribonucleotide synthetase (EC6.3.4.13, PurD) is AW169_RS11355.

[0047] Unless otherwise specified, other materials, reagents, etc. used in the examples of the present invention are commercially available.

[0048] During the fermentation process of producing nucleotide products, increasing the gene expression of key enzymes in the metabolic pathway, i.e., the de novo nucleotide synthesis pathway, is one of the important methods to increase the titer of the final product. Among them, glycinamide nucleotide synthetase (purD gene) is the second key enzyme in the de novo synthesis pathway, and its expression can be increased by promoter engineering. At present, the main industrial strain used in industrial fermentation production of nucleotide products is Corynebacterium stagnantum, but the strong constitutive promoters available in Corynebacterium stagnantum are very limited. Therefore, the present invention constructs a promoter library to screen out stronger promoters. Figure 2 In order to improve the compatibility of the promoter with the glycinamide nucleotide synthetase gene, the present invention uses the original promoter in the glycinamide nucleotide synthetase gene genome (nucleotide sequence as shown in SEQ ID NO: 2, named PpurD) as a template to construct a promoter library, uses sfGFP as a reporter protein, and adds a 180 bp sequence at the 5' end of the glycinamide nucleotide synthetase gene through a GGGGS linker in front of the reporter protein sfGFP. The strong promoter screened in this way can theoretically significantly increase the expression of the glycinamide nucleotide synthetase gene.

[0049] The present invention is further described below in conjunction with specific embodiments.

[0050] Example 1: Preparation of promoter mutants

[0051] (1) Construction of promoter strength characterization plasmid

[0052] Using the Escherichia coli-Corynebacterium shuttle plasmid pEC-XK99E as a template, primers pEC-XK99E1-S and pEC-XK99E1-A were designed to amplify the plasmid backbone DNA fragment; using pSenPutsfGFP as a template, sfGFP-S and sfGFP-A were used as primers to amplify the sfGFP fragment; the amplified plasmid backbone DNA fragment and the sfGFP fragment were connected by a one-step recombination cloning kit to obtain the pEC-XK99E-sfGFP recombinant plasmid. The recombinant plasmid pEC-XK99E-sfGFP was used as a template and pEC-XK99E-S and pEC-XK99E-A were used as primers to amplify the DNA fragments of the plasmid backbone and the superfolded green fluorescent protein gene; the genome of Corynebacterium stationis ATCC 6872 was used as a template and purD-S and purD-A were used as primers to amplify the DNA fragment of the PpurD promoter (the nucleotide sequence was shown in SEQ ID NO: 2) and the first 180 bp of the purD gene; the two fragments were connected by a one-step recombination cloning kit to obtain the pEC-PpurD-sfGFP recombinant plasmid.

[0053] The recombinant plasmid pEC-PpurD-sfGFP was used as a template and pEC-XK99E2-A and pEC-XK99E-purD-S were used as primers to amplify the vector fragment, which was then recombined and cloned with the Psod fragment amplified from Psod-S and Psod-purD-A and the Peftu fragment amplified from Peftu-S and Peftu-purD-A to obtain the recombinant plasmids pEC-Psod-sfGFP and pEC-Peftu-sfGFP. The primer sequences of the above primers are shown in Table 1.

[0054] Table 1 Primer sequence list

[0055]

[0056] (2) Preparation of transformation strains

[0057] The plasmids (vectors) pEC-PpurD-sfGFP, pEC-Psod-sfGFP and pEC-Peftu-sfGFP and pEC-XK99E prepared in step (1) were transformed into Corynebacterium stationis ATCC 6872 competent cells (Corynebacterium stationis ATCC 6872) by electroporation, and the following recombinant strains were obtained on a BHISG solid plate containing 25 μg / mL kanamycin: Corynebacterium stationis ATCC 6872 / pEC-PpurD-sfGFP, Corynebacterium stationis ATCC 6872 / pEC-Psod-sfGFP, Corynebacterium stationis ATCC 6872 / pEC-Peftu-sfGFP and Corynebacterium stationis ATCC 6872 / pEC-XK99E.

[0058] (3) Construction and screening of promoter library

[0059] The recombinant plasmid pEC-PpurD-sfGFP prepared in step (1) was used as a template, and the promoter fragments were respectively amplified using M-purD-S-1, M-purD-S-2, M-purD-S-3 and M-promoter-A containing mutant bases shown in Table 2 as primers, and the plasmid backbone was amplified using M-XK99E-S and M-XK99E-purD-A as primers. The promoter fragments were respectively connected to the plasmid backbone through 2 to 3 tubes of a one-step recombination cloning kit, each tube having 10 μL, to obtain the recombinant plasmids and transform Escherichia coli TOP10 competent cells, and the cells were spread on a LB solid culture medium containing 50 μg / mL kanamycin, and the total volume of the collection was about 10 6 The recombinant plasmid was extracted from the bacterial cells of the single clones to obtain the PpurD promoter library plasmid.

[0060] About 1 μg of the library plasmid was transferred into the competent cells of Corynebacterium ATCC 6872 by electroporation, and the cells were spread on about 20 BHISG solid plates containing 25 μg / mL kanamycin, each plate containing about 400-700 clones, and a total of about 10,000 clones were obtained. All clones on the plate were washed with PBS buffer for flow cytometry sorting (FACS), and clones with enhanced fluorescence were sorted and spread on BHISG solid plates containing 25 μg / mL kanamycin to obtain recombinant strains with enhanced fluorescence intensity.

[0061] Table 2 Primer sequence list

[0062]

[0063] (4) Strength characterization of promoter library

[0064] The recombinant strain with enhanced fluorescence intensity obtained by screening in step (3) and the recombinant strain constructed in step (2) were inoculated into a 48-deep-well plate, each well containing 800 μL of fermentation medium containing 25 μg / mL kanamycin, and cultured at 30°C and 800 r / min for 24 h. The plates were then transferred to a new 48-deep-well plate, each well containing 900 μL of fermentation medium containing 25 μg / mL kanamycin. Each sample was cultured in triplicate at 30°C and 800 r / min for 24 h. The OD was measured. 600 and fluorescence value (excitation wavelength is 488nm, emission wavelength is 520nm), and the fluorescence intensity is the measured fluorescence value / OD 600 , i.e. GFU / OD 600 .

[0065] See Table 3 and Figure 3 , GFU / OD 600 The promoter mutant with the highest value was named PpurDmut, and its corresponding recombinant strain was named ATCC 6872 / pEC-PpurDmut-sfGFP. The recombinant strain exhibited at least 33 times the fluorescence intensity of ATCC 6872 / pEC-PpurD-sfGFP (i.e., the recombinant strain of the original promoter), and the promoter mutant PpurDmut also showed higher fluorescence sensitivity than Psod and Peftu, which are known to be strong promoters.

[0066] Table 3 Fluorescence intensity of stagnant Corynebacterium

[0067]

[0068] PCR amplification of ATCC 6872 / pEC-PpurDmut-sfGFP single colony was performed using primers Promoter-CX-S and Promoter-CX-A shown in Table 4, and the amplified PCR fragment was sequenced. The results showed that the nucleotide sequence of PpurDmut was SEQ ID NO: 1, which differed from the original promoter PpurD in that the nucleotides at positions 40, 42, 44 to 51, and 53 to 56 of PpurDmut (sequence shown in SEQ ID NO: 1) were mutated from C, G, CGAGCGAA and AAGT at the corresponding positions of PpurD (sequence shown in SEQ ID NO: 2) to A, A, GCCCAATT and TCTG.

[0069] Table 4 Primer sequence list

[0070]

[0071] Example 2: Verification of promoter mutant efficacy

[0072] (1) Construction of recombinant strains

[0073] The glycinamide nucleotide synthetase gene (purD) of Corynebacterium stagnantum ATCC 6872 was used as a reporter protein to construct recombinant vectors pEC-PpurD-purD, pEC-Psod-purD and pEC-PpurDmut-purD. The recombinant vectors were electroporated into competent cells of Corynebacterium stagnantum ATCC 6872 to construct corresponding recombinant strains: Corynebacterium stagnantum ATCC 6872 / pEC-PpurD-purD, Corynebacterium stagnantum ATCC 6872 / pEC-Psod-purD and Corynebacterium stagnantum ATCC 6872 / pEC-PpurDmut-purD.

[0074] (2) Extraction of total RNA

[0075] The recombinant strains ATCC 6872 / pEC-PpurD-purD, ATCC 6872 / pEC-Psod-purD and ATCC6872 / pEC-PpurDmut-purD were inoculated into LB seed medium and cultured at 30°C and 250 rpm for 20 h to obtain seed solution. 600 The concentration of 1% iodine was 1, and the seed solution was transferred to the basic culture medium and cultured for 8 h. 4 mL of the fermentation solution of each strain was taken and centrifuged at 6000 rpm / min for 3 min, and the supernatant was discarded to collect the bacteria.

[0076] Add 1.2 mL of AES-phenol-chloroform mixture to each cell to fully resuspend the cells, shake vigorously, and treat at 65°C for 15 minutes, shaking for 3 minutes and resting for 3 minutes. Then put in an ice bath for 5 minutes, centrifuge at 4°C and 12000g for 10 minutes, carefully transfer the upper aqueous phase to a centrifuge tube containing 600 μL chloroform, mix, centrifuge at 4°C and 12000g for 10 minutes, carefully transfer the upper aqueous phase to a clean 1.5 mL centrifuge tube, add 2.5 times the volume of anhydrous ethanol and 1 / 10 of 3M NaAc, turn the centrifuge tube upside down 2 to 3 times, and place it at -20°C for precipitation for 30 minutes. Centrifuge at 4°C and 12000g for 15 minutes, discard the supernatant, wash the precipitate with 1 mL of 75% ethanol, centrifuge at 4°C and 12000g for 5 minutes, discard the supernatant, centrifuge briefly and aspirate the remaining liquid in the centrifuge tube. The precipitate was placed on a clean bench for 2 to 3 minutes, 20 μL of RNase-free water was added to dissolve the RNA precipitate, 1 μL of RNA solution was taken and the concentration of the RNA solution was detected by Nanodrop (Thermo), and the A260 / A280 value was recorded. If the value was between 1.8 and 2.0, the RNA quality was considered qualified, and the RNA was stored at -80°C and used in subsequent experiments.

[0077] (3) Removal of DNA from RNA samples and reverse transcription

[0078] The extraction of total RNA may be contaminated with genomic DNA, so the genomic DNA in RNA is removed before reverse transcription. The system for removing gDNA shown in Table 5 is placed in a PCR instrument for reaction, and the reaction program is: 42°C, 2min, and incubated at 4°C.

[0079] Table 5 System for removing gDNA

[0080]

[0081] The system after the above reaction was subjected to reverse transcription reaction: the reverse transcription PCR system shown in Table 6 was placed in a PCR instrument for reaction; the reaction program was: heating at 37°C for 15 min, heating at 85°C for 5 sec, and keeping warm at 4°C.

[0082] Table 6 Reverse transcription PCR system

[0083]

[0084] (4) Fluorescence quantitative PCR

[0085] Fluorescence quantitative PCR uses ABI's Q1 fluorescence quantitative PCR instrument and Acryl Bio's SYBR Green Pro Taq HS premixed qPCR kit (containing tracer dye and Rox). 20 μL of fluorescence quantitative PCR reaction system is added to a 96-well plate and placed in the fluorescence quantitative PCR instrument for reaction. The PCR reaction system is shown in Table 7, the PCR reaction program is shown in Table 8, and the primers used in PCR are shown in Table 9.

[0086] Table 7 Fluorescence quantitative PCR reaction system

[0087]

[0088] Table 8 PCR reaction program

[0089]

[0090] Table 9 Fluorescence quantitative PCR primer sequence list

[0091]

[0092] 16S rRNA was selected as the internal reference gene, and the transcription levels of other related genes were measured using 2 -△△Ct Method calculation.

[0093] The results of fluorescence quantitative PCR were as follows Figure 4As shown, after 24 hours of fermentation, the transcription level of the purD gene in the recombinant strain ATCC 6872 / pEC-PpurDmut-purD was significantly increased compared with the recombinant strain ATCC 6872 / pEC-PpurD-purD of the original promoter, proving that the promoter mutant PpurDmut can significantly increase the transcription level of the purD gene in Corynebacterium stagnantum, thereby increasing the expression of glycinamide nucleotide synthetase. Glycinamide nucleotide synthetase is a key enzyme in the nucleotide synthesis metabolic pathway. Increased expression of glycinamide nucleotide synthetase can increase the metabolic flux of nucleotide products, thereby increasing the yield of nucleotides.

[0094] Example 3: Evaluation of the ability of promoter mutants to express heterologous α-amylase genes

[0095] (1) Construction of recombinant strains

[0096] Recombinant vectors pEC-PpurD-amyE, pEC-Psod-amyE and pEC-PpurDmut-amyE were constructed using α-amylase amyE gene (from Bacillus subtilis subsp. subtilis str.168) as a reporter protein. The pEC-PpurD-amyE, pEC-Psod-amyE and pEC-PpurDmut-amyE recombinant vectors were electroporated into the competent cells of Corynebacterium stagnantum ATCC 6872 to construct the corresponding recombinant strains ATCC 6872 / pEC-PpurD-amyE, ATCC 6872 / pEC-Psod-amyE and ATCC 6872 / pEC-PpurDmut-amyE.

[0097] (2) Inducible expression of α-amylase activity

[0098] The recombinant strains ATCC 6872 / pEC-PpurD-amyE, ATCC 6872 / pEC-Psod-amyE and ATCC6872 / pEC-PpurDmut-amyE were cultured in seed solution for 20 h, and the initial OD 600=0.1 and transferred it into the fermentation medium. After 24 hours of fermentation, the cells were collected, 1 mL of the fermentation liquid was collected in a 2 mL centrifuge tube, centrifuged at 7000 rpm for 3 min, washed with PBS buffer (pH 7.4) 3 times, resuspended with 1 mL of PBS buffer (pH 7.4) and transferred to a crushing tube containing 0.5 g of zirconium oxide beads, and crushed using a Bead Ruptor 12 (Omni International, Inc., USA) instrument. Each lysis was 30 s, placed on ice for 1 min, and the operation was repeated for 10 cycles. The sample was centrifuged at 4 ° C, 14000 rpm for 3 min, and the supernatant was slowly aspirated for subsequent protein concentration determination and α-amylase activity determination.

[0099] (3) α-amylase activity detection

[0100] The protein concentration was determined by the Bradford method, and the amylase activity was determined using the α-amylase (α-AL) activity detection kit of Beijing Box Biotechnology Co., Ltd. The determination method is as follows:

[0101] Dilute the 10 mg / mL glucose standard solution with distilled water to 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, and 0.00625 mg / mL to obtain a standard dilution. Dilute the supernatant in step (2) by an appropriate multiple to obtain an amylase stock solution. Take an appropriate amount of the amylase stock solution and treat it in a boiling water bath for 5 minutes (seal to prevent water loss), cool to room temperature, and obtain an inactivated enzyme stock solution.

[0102] The determination system is shown in Table 10. 250 μL amylase stock solution, 250 μL inactivated enzyme stock solution, 250 μL standard diluent and 250 μL distilled water are added to the determination tube, control tube, standard tube and blank tube respectively. Then, each system is fully mixed, placed at 70°C for 15 minutes, cooled to room temperature, and then 250 μL reagent 2 is added to the determination tube. Each system is fully mixed and reacted accurately at 40°C for 5 minutes. Then 500 μL reagent 1 is added to the determination tube, and 500 μL reagent 1 and 250 μL reagent 2 are added to the control tube, standard tube and blank tube respectively. Each system is mixed evenly, treated in a boiling water bath for 10 minutes and then cooled to room temperature. The reaction solution is taken to a 96-well ELISA plate, and the absorbance at 540 nm is measured in an ELISA instrument, recorded as A determination, A control, A standard and A blank; ΔA determination = A determination-A control, ΔA standard = A standard-A blank are calculated. Among them, a control tube is required for each determination tube.

[0103] Table 10 α-amylase activity assay system

[0104]

[0105]

[0106] A standard curve was drawn with the standard dilution concentrations of 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, and 0.00625 mg / mL as the abscissa (x) and the corresponding ΔA standard as the ordinate (y) to obtain the linear regression equation y=kx+b. The ΔA determination was substituted into the formula to obtain x (mg / mL).

[0107] The enzyme activity unit of α-amylase (α-AL) is defined as: one unit of enzyme activity is the amount of reducing sugar produced by one mg of tissue protein per minute.

[0108] The calculation formula of α-amylase (α-AL) activity is: Where Vsample is the volume of enzyme solution added to the reaction system, i.e. 0.25 mL; Cpr (mg / mL) is the sample protein concentration; T is the reaction time, i.e. 5 min.

[0109] The x and Cpr measured for each recombinant bacterium were substituted into the above enzyme activity calculation formula, and the calculated enzyme activity is shown in Table 11.

[0110] Table 11 α-amylase activity in each recombinant strain of ATCC 6872

[0111]

[0112] Referring to Table 11, the α-amylase amyE gene from Bacillus subtilis subsp. subtilis str.168 can be heterologously expressed in Corynebacterium stationis, and compared with Corynebacterium stationis ATCC6872 / pEC-PpurD-amyE and Corynebacterium stationis ATCC 6872 / pEC-PpurDmut-amyE, Corynebacterium stationis ATCC6872 / pEC-PpurDmut-amyE exhibited significantly increased α-amylase activity, indicating that PpurDmut is a universal and potent promoter.

[0113] In summary, compared with the original promoter PpurD and the existing strong promoter Psod, the promoter mutant PpurDmut provided by the present invention can enhance the transcription level of glycinamide nucleotide synthetase, thereby promoting the expression of glycinamide nucleotide synthetase in Corynebacterium stagnation, and thus can increase the metabolic flux of nucleotide products. In addition, the promoter of the present invention can increase the expression of heterologous genes (such as α-amylase amyE gene from Bacillus subtilis subsp.subtilis str.168) in Corynebacterium stagnation, and achieves α-amylase activity that is 2.30 times higher than the original promoter PpurD and 2.24 times higher than the existing strong promoter Psod, so the promoter provided by the present invention can be used as a universal promoter and has strong industrial applicability.

[0114] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

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

1.

2. A gene expression cassette, characterized in that: It comprises the promoter as claimed in claim 1, and a target gene operably linked to the promoter.

3. A recombinant vector, characterized in that: It comprises the promoter according to claim 1, or the gene expression cassette according to claim 2.

4. A recombinant strain, characterized in that: Comprising the gene expression cassette as claimed in claim 2, or the recombinant vector as claimed in claim 3.

5. The recombinant strain according to claim 4, characterized in that The host cell of the recombinant strain is Corynebacterium stasis.

6. The recombinant strain according to claim 5, characterized in that The stagnant bacterium is stagnant bacterium ATCC 6872.

7. Use of the promoter according to claim 1 in promoting efficient expression of a target gene in Corynebacterium stagnans.

8. A method for producing nucleotide products, characterized in that: The method comprises culturing a recombinant strain containing the promoter of claim 1 operably linked to a glycinamide nucleotide synthetase gene, and collecting the produced nucleotides.

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