Promoter, recombinant microorganism producing threonine and use thereof

Abstract

The present invention relates to the field of microbial engineering technology, specifically to a promoter, a recombinant microorganism producing threonine, and its use. The present invention provides a promoter having the nucleotide sequence shown in SEQ ID NO.1. The present invention provides a mutant dapA gene promoter with significantly reduced transcription activity by rationally designing the dapA gene promoter, which is used for weakened expression of microbial genes. By replacing the original promoters of the dapA gene, ddh gene, ilvA gene, tdcB gene, and gltA gene in Corynebacterium glutamicum with this promoter, the expression levels of these genes are significantly reduced, and in the recombinant microorganism constructed thereby, the carbon metabolic flow is more diverted to the threonine synthesis metabolic pathway, the threonine production ability of the strain is significantly improved, the threonine production amount is significantly improved compared to the starting strain, and the growth performance is good.

Description

[Technical field]

[0001] The present invention relates to the field of microbial engineering technology, and more particularly to a promoter, a recombinant microorganism that produces threonine, and its use. [Background technology]

[0002] L-Threonine has the chemical name β-hydroxy-α-aminobutyric acid, the molecular formula C4H9NO3, and the relative molecular mass 119.12. L-Threonine is an essential amino acid, and is mainly used in medicines, chemical drugs, food supplements, feed additives, etc.

[0003] In Corynebacterium glutamicum, the production of threonine from oxaloacetate requires five catalytic steps, and the catalytic enzymes involved in these five steps are aspartokinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), homoserine dehydrogenase (encoded by hom), homoserine kinase (encoded by thrB), and threonine synthase (encoded by thrC). Concerning the lysC gene and the hom gene, the hom gene and the lysC gene that are resistant to feedback inhibition have been reported (Reinscheid DJ, Eikmanns BJ, Sahm H. Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J]. Journal of Bacteriology, 1991, 173(10):3228-3230; Eikmanns BJ, Eggeling L, Sahm H. Molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum.[J]. Antonie Van Leeuwenhoek, 1993, 64(2):145-163.).At present, there are several reports on the construction of engineered bacteria that produce threonine by fermentation using Corynebacterium glutamicum. For example, Lothar Eggling et al. increased the amount of threonine produced from 49 mM to 67 mM by weakening the coding gene glyA in the threonine utilization pathway and overexpressing the threonine exportin ThrE (Simic P, Willuhn J, Sahm H, et al. Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase L-Threonine Accumulation by Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2002, 68(7):3321-3327.).

[0004] Currently, reports on threonine production by Corynebacterium glutamicum have focused mainly on metabolic engineering modification of the threonine synthesis pathway, but there are few reports on the optimization of carbon metabolic flow. In addition, when controlling gene expression in Corynebacterium glutamicum, gene expression is mainly enhanced by introducing artificially synthesized strong promoters, strong promoters of endogenous genes, or strong heterologous promoters, but there are relatively few reports on promoters that weaken gene expression, and gene weakening mainly relies on start codon replacement and gene knockout techniques. Summary of the Invention

[0005] An object of the present invention is to provide a mutant dapA gene promoter. Another object of the present invention is to provide a recombinant microorganism constructed by controlling gene expression with this promoter, and use thereof.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] The present invention provides a promoter having the nucleotide sequence shown in SEQ ID NO.1.

[0008] The promoter is a mutant dapA gene (4-hydroxytetrahydrodipicolinate synthase gene) promoter (mPdapA), and this mutant promoter has the activity of initiating gene transcription, but the activity of the promoter to initiate gene transcription is significantly reduced compared to the wild-type dapA gene promoter.

[0009] Preferably, the nucleotide sequence of said mutant dapA gene promoter is shown in SEQ ID NO.1.

[0010] The present invention also provides a biological material, which comprises the mutant dapA gene promoter, and is a recombinant DNA, vector or host cell.

[0011] The recombinant DNA may be recombinant DNA in which a gene of interest is operably linked downstream of the promoter, or recombinant DNA in which a gene of interest is operably linked downstream of the promoter and another transcription control element or translation control element is operably linked upstream or downstream of the promoter, or recombinant DNA in which a homology arm fragment for homologous recombination is operably linked upstream and / or downstream of the promoter.

[0012] The vector may be a plasmid vector, a viral vector or a transposon.

[0013] The host cell is preferably a microbial cell.

[0014] The microbial cells are preferably bacteria of the genus Escherichia or Corynebacterium. Among them, the bacteria of the genus Escherichia are preferably Escherichia coli, and the bacteria of the genus Corynebacterium are preferably Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, or Corynebacterium aminogenes.

[0015] The present invention provides the use of said mutant dapA gene promoter for initiating expression of a gene of interest.

[0016] Specifically, the use involves operably linking a target gene with a mutant dapA gene promoter to obtain a recombinant DNA, and then introducing the recombinant DNA into a host cell to express the target gene.

[0017] The mutant dapA gene promoter is used to drive the expression of metabolism-related genes of a target metabolic product, in particular to reduce the expression of genes related to competing pathways of the target metabolic product and improve the accumulation of the target metabolic product.

[0018] The present invention also provides a use of the mutant dapA gene promoter for improving the production amount or conversion rate of a microbial metabolic product, or for constructing a strain producing the microbial metabolic product.

[0019] Preferably, the metabolic product is threonine or a derivative thereof.

[0020] In the threonine fermentation production process, lysine and isoleucine are the main by-products of threonine fermentation production, and the lysine and isoleucine synthesis pathway is the main competing pathway of the threonine synthesis pathway. Therefore, it is necessary to block or weaken the by-product pathway such as the lysine synthesis pathway, and to allow more carbon metabolic flow to flow to the threonine synthesis pathway. However, directly knocking out genes such as dapA, which is a lysine synthesis gene, leads to a rapid decrease in the growth rate of the strain and nutrient requirement of the cells, which is disadvantageous to threonine fermentation production. The present invention has found that by weakening the lysine and isoleucine synthesis genes with the mutant dapA gene promoter, it is possible to avoid nutrient requirement, ensure normal growth of the strain, and significantly improve the distribution of carbon metabolic flow to the threonine synthesis pathway, enhance the threonine synthesis ability of the strain, and reduce the accumulation of lysine and isoleucine.

[0021] Based on the above findings, the present invention provides a recombinant microorganism in which a gene encoding at least one enzyme selected from the following (1) to (4) is transcriptionally driven by the mutant dapA gene promoter: (1) 4-Hydroxytetrahydrodipicolinate synthase (2) Diaminopimelate dehydrogenase (3) Threonine dehydratase (4) Citrate synthase

[0022] In one embodiment of the present invention, in the recombinant microorganism, genes encoding 4-hydroxytetrahydrodipicolinate synthase and diaminopimelate dehydrogenase are transcriptionally driven by the mutant dapA gene promoter.

[0023] In another embodiment of the present invention, in the recombinant microorganism, genes encoding 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, and threonine dehydratase are transcriptionally driven by the mutant dapA gene promoter.

[0024] In another embodiment of the present invention, in the recombinant microorganism, the genes encoding 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase and citrate synthase are all transcriptionally driven by the mutant dapA gene promoter.

[0025] The 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase and citrate synthase have NCBI reference sequence numbers WP_011014792.1, WP_011015254.1, WP_011014022.1 and WP_003862033.1, respectively, or are amino acid sequences having 90% identity to the reference sequences and equivalent functions.

[0026] Specifically, being transcriptionally driven by the mutant dapA gene promoter means replacing the original promoter of the gene with the mutant dapA gene promoter.

[0027] The expression levels of 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase, and citrate synthase in the recombinant microorganism are significantly reduced, the ability to synthesize threonine and its derivatives is significantly enhanced, and compared with the starting strain, the threonine production and conversion rate are significantly higher, and the accumulation of by-products is significantly reduced.

[0028] Since microorganisms have strict metabolic control, aspartokinase and homoserine dehydrogenase in the threonine synthesis pathway are strictly controlled by the intracellular threonine concentration. Therefore, for the starting strain capable of accumulating threonine, it is necessary to first penetrate the synthesis pathway, which mainly involves removing the feedback inhibition of aspartokinase and homoserine dehydrogenase, enhancing the enzyme activity, etc.

[0029] Specifically, in the recombinant microorganism, the enzyme activities of aspartokinase and / or homoserine dehydrogenase are enhanced and / or feedback inhibition is relieved. Aspartokinase and homoserine dehydrogenase have the reference sequence numbers WP_003855724.1 and WP_003854900.1 on NCBI, respectively, or are amino acid sequences having 90% identity to the above reference sequences and having equivalent functions.

[0030] Preferably, the microorganism is one of the following: 1 )~( 7 ) ( 1 A microorganism in which a gene encoding 4-hydroxytetrahydrodipicolinate synthase is transcriptionally driven by the mutant dapA gene promoter, and in which the enzyme activities of aspartokinase and / or homoserine dehydrogenase are enhanced and / or feedback inhibition is relieved. ( 2 A microorganism in which a gene encoding diaminopimelate dehydrogenase is transcriptionally driven by the mutant dapA gene promoter, and in which the enzyme activities of aspartokinase and / or homoserine dehydrogenase are enhanced and / or feedback inhibition is relieved. ( 3 A microorganism in which a gene encoding threonine dehydratase is transcriptionally driven by the mutant dapA gene promoter, and in which the enzyme activities of aspartokinase and / or homoserine dehydrogenase are enhanced and / or feedback inhibition is relieved. ( 4 A microorganism in which a gene encoding citrate synthase is transcriptionally driven by the mutant dapA gene promoter, and in which the enzyme activities of aspartokinase and / or homoserine dehydrogenase are enhanced and / or feedback inhibition is relieved. ( 5A microorganism in which genes encoding 4-hydroxytetrahydrodipicolinate synthase and diaminopimelate dehydrogenase are transcriptionally driven by the mutant dapA gene promoter, and the enzyme activities of aspartokinase and / or homoserine dehydrogenase are enhanced and / or feedback inhibition is relieved. ( 6 A microorganism in which genes encoding 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, and threonine dehydratase are transcriptionally driven by the mutant dapA gene promoter, and the enzyme activities of aspartokinase and / or homoserine dehydrogenase are enhanced and / or feedback inhibition is relieved. ( 7 A microorganism in which genes encoding 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase, and citrate synthase are transcriptionally driven by the mutant dapA gene promoter, and the enzyme activities of aspartokinase and / or homoserine dehydrogenase are enhanced and / or feedback inhibition is relieved.

[0031] Preferably, the enhancement of the enzyme activity is achieved by the following 1) to 6) or any combination thereof. 1) Enhancement by introducing a plasmid carrying a gene encoding the enzyme 2) Enhancement by increasing the copy number of the gene encoding the enzyme in the chromosome. 3) Enhancement by modifying the promoter sequence of the gene encoding the enzyme in the chromosome. 4) Enhancement by operably linking a strong promoter to the gene encoding the enzyme. 5) Enhancing the enzyme by modifying its amino acid sequence 6) Enhancement by modifying the nucleotide sequence encoding the enzyme

[0032] In Corynebacterium glutamicum, the threonine dehydratase is encoded by the ilvA and tdcB genes.

[0033] More preferably, the enhancement of the enzyme activity is achieved by replacing the original promoter of the gene encoding the enzyme with a strong promoter.

[0034] Preferably, the strong promoter is Psod or PcspB.

[0035] Here, the enzymatic activity of aspartokinase is preferably enhanced by replacing the original promoter of the coding gene with the strong promoter Psod and mutating the initiation codon to ATG.

[0036] Preferably, the enhancement of the enzymatic activity of homoserine dehydrogenase is achieved by replacing the original promoter of the encoding gene with the strong promoter PcspB.

[0037] De-inhibition of aspartokinase feedback is preferably achieved by generating a T311I mutation.

[0038] The release of feedback inhibition of homoserine dehydrogenase is preferably achieved by generating a G378E mutation.

[0039] The recombinant microorganism according to the present invention is preferably a bacterium of the genus Corynebacterium, more preferably Corynebacterium glutamicum, including ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum phylogenetic tree https: / / www.ncbi.nlm.nih.gov / genome / 469), and more preferably Corynebacterium glutamicum ATCC 13032.

[0040] The present invention also provides a method for constructing the recombinant microorganism, which comprises replacing a promoter of a gene encoding at least one of the enzymes 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase, and citrate synthase with the mutant dapA gene promoter.

[0041] Preferably, the method further comprises enhancing the enzymatic activity of aspartokinase and / or homoserine dehydrogenase and / or relieving feedback inhibition.

[0042] Here, the enhancement of the enzyme activity is achieved by the following 1) to 6) or any combination thereof. 1) Enhancement by introducing a plasmid carrying a gene encoding the enzyme 2) Enhancement by increasing the copy number of the gene encoding the enzyme in the chromosome. 3) Enhancement by modifying the promoter sequence of the gene encoding the enzyme in the chromosome. 4) Enhancement by operably linking a strong promoter to the gene encoding the enzyme. 5) Enhancing the enzyme by modifying its amino acid sequence 6) Enhancement by modifying the nucleotide sequence encoding the enzyme

[0043] The modification methods, including gene enhancement, for the above strains are all known to those skilled in the art, and can be seen in the following: Man Zaiwei, Systematic Pathway Engineering Modification of Corynebacterium crenatum with High L-Arginine Production [D], Jiangnan University, 2016; Cui Yi, Metabolic Engineering Modification of Corynebacterium glutamicum for L-Leucine Production [D], Tianjin University of Science and Technology; Xu Guodong, Construction of a Strain Producing L-Isoleucine and Optimization of Fermentation Conditions, Tianjin University of Science and Technology, 2015.

[0044] The present invention also provides any one of the following uses of the recombinant microorganism: (1) Use for fermentation production of microbial metabolic products or their derivatives (2) Use for selecting strains that produce microbial metabolic products or their derivatives (3) Use for improving the production and / or conversion rate of microbial metabolic products

[0045] Preferably, the microbial metabolic product is threonine.

[0046] The present invention also provides a method for producing threonine or a derivative thereof by fermentation, which comprises the steps of culturing the recombinant microorganism and isolating and obtaining threonine or a derivative thereof from the culture.

[0047] Specifically, the method includes inoculating the recombinant microorganism into a seed medium to perform seed culture, obtaining a seed liquid, inoculating the seed liquid into a fermentation medium to perform culture, obtaining a fermentation liquid, and separating and extracting the fermentation liquid to obtain threonine or a derivative thereof.

[0048] Preferably, the fermentation medium contains 45-55 mL / L corn steep liquor, 25-35 g / L glucose, 3-5 g / L ammonium sulfate, 25-35 g / L MOPS, 8-12 g / L potassium dihydrogen phosphate, 15-25 g / L urea, 8-12 mg / L biotin, 5-7 g / L magnesium sulfate, 0.5-1.5 g / L ferrous sulfate, 15-25 g / L urea, 15-25 g / L urea, 15-25 mg ... B1·It contains HCl 35-45 mg / L, calcium pantothenate 45-55 mg / L, nicotinamide 35-45 mg / L, manganese sulfate 0.5-1.5 g / L, zinc sulfate 15-25 mg / L, and copper sulfate 15-25 mg / L, and has a pH of 7.0-7.2.

[0049] The present invention has the following beneficial effects: By rationally designing the promoter of the dapA gene, the present invention obtains a mutant dapA gene promoter with significantly reduced transcription activity, and this promoter is used for attenuated expression of a microbial gene.

[0050] By replacing the original promoters of the genes encoding 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase, and citrate synthase in Corynebacterium glutamicum with this promoter, the expression levels of these genes were significantly reduced. As a result, in the recombinant microorganism constructed as a result, more carbon metabolic flow was directed to the threonine synthesis metabolic pathway, significantly improving the threonine production ability of the strain, with the threonine production amount being 279% higher than that of the starting strain, and the strain having good growth performance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The following examples are intended to illustrate the present invention and are not intended to limit the scope of the invention.

[0052] Details of the genes and enzymes in the following examples are as follows: Aspartokinase: coding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247. Homoserine dehydrogenase: coding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136. 4-Hydroxytetrahydrodipicolinate synthase: coding gene name dapA, NCBI number: cg2161, Cgl1971, NCgl1896. Diaminopimelate dehydrogenase: coding gene name ddh, NCBI number: cg2900, Cgl2617, NCgl2528. Threonine dehydratase: coding gene name ilvA, NCBI number: cg2334, Cgl2127, NCgl2046. Threonine dehydratase: coding gene name tdcB, NCBI number: cg1116, Cgl0978, NCgl0939. Citrate synthase: coding gene name gltA, NCBI number: cg0949, Cgl0829, NCgl0795.

[0053] The present invention uses the wild-type Corynebacterium glutamicum ATCC13032 as a base to construct a threonine-producing strain through metabolic engineering modification. First, the threonine synthesis pathway is opened, mainly including the release of feedback inhibition and enhanced expression of aspartokinase (lysC) and homoserine dehydrogenase (hom). Specifically, based on Corynebacterium glutamicum ATCC13032, the expression of aspartokinase is enhanced and the feedback inhibition of the enzyme is released by mutation to obtain a modified strain SMCT136. Then, the expression of homoserine dehydrogenase is enhanced and the feedback inhibition of the enzyme is released by mutation to obtain a modified strain SMCT137. This strain has a rudimentary ability to synthesize threonine, and its threonine production is 2.4 g / L.

[0054] In the modified bacterium SMCT137, lysine and isoleucine are the main by-products of threonine fermentation production, and the lysine and isoleucine synthesis pathway is the main competing pathway for the threonine synthesis pathway. Therefore, it is necessary to block or weaken the by-product pathway such as the lysine synthesis pathway and to allow more carbon metabolic flow to the threonine synthesis pathway. However, directly knocking out the lysine synthesis gene such as dapA leads to a rapid decrease in the growth rate of the strain and nutrient requirement of the cells, which is disadvantageous for threonine fermentation production. For this reason, the present invention adopts a method for rationally modifying the dapA gene promoter, and obtains a mutant dapA gene promoter (mPdapA) through rational design and selection, which is expected to reduce the expression level of 4-hydroxytetrahydrodipicolinate synthase, thereby achieving the purpose of balancing cell growth and carbon flow optimization. Based on SMCT137, the present invention uses site-specific mutation (replace the original dapA gene promoter with the mutant dapA promoter) in the promoter of 4-hydroxytetrahydrodipicolinate synthase coding gene dapA in the lysine synthesis pathway to obtain SMCT138 strain, in which the expression level of 4-hydroxytetrahydrodipicolinate synthase is reduced, the threonine production ability is improved from 2.4 g / L to 3.0 g / L, and the content of the by-product lysine is reduced from 1.1 g / L to 0.8 g / L.

[0055] Furthermore, in the present invention, the promoters of ddh, ilvA, tdcB, and gltA are replaced with the mutant dapA promoter using SMCT137 as the starting strain to obtain modified strains SMCT139, SMCT140, SMCT141, and SMCT142. It is confirmed that the expression levels of the corresponding proteins, diaminopimelate dehydrogenase, threonine dehydratase (ilvA), threonine dehydratase (tdcB), and citrate synthase, are reduced; by replacing the ddh promoter, the lysine content is reduced from 1.1 g / L to 0.9 g / L, and the threonine production is increased by 0.4 g / L; by replacing the ilvA and tdcB promoters, the isoleucine content is reduced by 0.6 g / L and 0.2 g / L, respectively, and the threonine production is increased by 0.8 g / L and 0.6 g / L, respectively; and after replacing the gltA promoter, the threonine production is increased by 0.8 g / L.

[0056] Furthermore, by incorporating the site-specific modifications stepwise into SMCT138 after the individual weakening, modified strains SMCT143, SMCT144, SMCT145, and SMCT146 are obtained. In the modified strain SMCT143, the amount of threonine produced is increased by 20%, and the by-product lysine is reduced from 0.8 g / L to 0.4 g / L, in the modified strain SMCT144, the amount of threonine produced is increased by 67%, and the by-product isoleucine is reduced from 1.3 g / L to 0.6 g / L, in the modified strain SMCT145, the amount of threonine produced is increased by 113%, and the by-product isoleucine is reduced from 0.6 g / L to 0.3 g / L, and in the modified strain SMCT146, the amount of threonine produced is increased by 203%. EXAMPLES

[0057] The construction process of the modified strain and the threonine fermentation production performance of the strain will be specifically described below with reference to examples.

[0058] Example 1 Construction of a plasmid for modifying the genome of a strain

[0059] 1. Aspartokinase expression enhancing plasmid pK18mobsacB-Psod-lysC g1a-T311I Construction Using the ATCC13032 genome as a template, PCR amplification was performed with the P21 / P22 primer set to obtain the upstream homologous arm up, PCR amplification was performed with the P23 / P24 primer set to obtain the promoter fragment Psod, and PCR amplification was performed with the P25 / P26 primer set to obtain lysC g1a-T311I The downstream homologous arm dn was obtained by PCR amplification using the P27 / P28 primer set. The up and Psod fragments were used as templates to perform fusion PCR using the P21 / P24 primer set to obtain the up-Psod fragment. g1a-T311I Fusion PCR was performed using dn as a template and the P21 / P28 primer set to obtain the full-length fragment up-Psod-lysC g1a-T311I pK18mobsacB was digested with BamHI / HindIII. The two were assembled using a seamless cloning kit, and Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-Psod-lysC. g1a-T311I was obtained.

[0060] 2. Homoserine dehydrogenase expression enhancing plasmid pK18mobsacB-PcspB-hom GapG378E Construction The plasmid construction method was as described in 1, and the primers used were P29, P30, P31, P32, P33, P34, P35, and P36.

[0061] 3. Construction of 4-hydroxytetrahydrodipicolinate synthase promoter replacement plasmid pK18mobsacB-ΔPdapA::mPdapA A dapA promoter with a mutated sequence, mPdapA (the sequence is shown in SEQ ID NO.1), was artificially synthesized. Using the artificially synthesized mPdapA sequence as a template, PCR amplification was performed with the PW21 / 22 primer set to obtain an mPdapA promoter fragment. Using the ATCC13032 genome as a template, PCR amplification was performed with the PW1 / 2 primer set to obtain the upstream homologous arm up, PCR amplification was performed with the PW3 / 4 primer set to obtain the upstream homologous arm dn, and using up, mPdapA, and dn as templates, fusion PCR was performed with the PW1 / 4 primer set to obtain the fragment up-mPdapA-dn. pK18mobsacB was cleaved with BamHI / HindIII. Both were assembled using a seamless cloning kit, and Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-ΔPdapA::mPdapA.

[0062] 4. Construction of diaminopimelate dehydrogenase promoter replacement plasmid pK18mobsacB-ΔPddh::mPdapA For the plasmid construction method, see 3, and the primers used were PW5, PW6, PW7, PW8, PW21, and PW22.

[0063] 5. Construction of threonine dehydratase (ilvA) promoter replacement plasmid pK18mobsacB-ΔPilvA::mPdapA For the plasmid construction method, see 3, and the primers used were PW9, PW10, PW11, PW12, PW21, and PW22.

[0064] 6. Construction of threonine dehydratase (tdcB) promoter replacement plasmid pK18mobsacB-ΔPtdcB::mPdapA For the plasmid construction method, see 3, and the primers used were PW13, PW14, PW15, PW16, PW21, and PW22.

[0065] 7. Construction of citrate synthase promoter replacement plasmid pK18mobsacB-ΔPgltA::mPdapA For the plasmid construction method, see 3, and the primers used were PW17, PW18, PW19, PW20, PW21, and PW22.

[0066] The primers used in the above plasmid construction process are shown in Table 1.

[0067] [Table 1]

[0068] Example 2 Construction of genome modified strain

[0069] 1. Construction of an aspartokinase enhanced expression strain ATCC13032 competent cells were prepared according to the classical method (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-Psod-lysC g1a-T311I transformed the competent cells by electroporation and selected transformants in which the target gene had been integrated into the chromosome by homology in a selective medium containing 15 mg / L kanamycin. The selected transformants were cultured overnight in a normal liquid brain-heart infusion medium, cultured at 30°C and shaken at 220 rpm in a rotary shaker. During this culture process, the transformants underwent a second recombination, and the vector sequence was removed from the genome by gene exchange. The culture was serially diluted (10 -2 From 10 -4The strain was serially diluted to 1000 μg / ml, and the dilutions were applied to a standard solid brain heart infusion medium containing 10% sucrose and incubated at 33°C for 48 h. The genome of the strain grown on the sucrose medium did not carry the integrated vector sequence. The target fragment was amplified by PCR, and the target mutant strain obtained by nucleotide sequencing analysis was named SMCT136. In this strain, the lysC gene was mutated, with the corresponding initiation codon mutated from GTG to ATG, the 311th amino acid encoded by it mutated from threonine to isoleucine, and the promoter of the lysC gene was replaced with the strong promoter Psod.

[0070] 2. Construction of homoserine dehydrogenase expression-enhanced strain The strain construction method was as described in 1. SMCT136 was used as the starting strain, and pK18mobsacB-PcspB-hom GapG378E was introduced into this starting strain to enhance homoserine dehydrogenase expression, and the obtained modified strain was named SMCT137. In this strain, the hom gene was mutated, the corresponding amino acid mutation site was G378E, and the promoter of the hom gene was replaced with the strong promoter PcspB.

[0071] 3. Construction of 4-hydroxytetrahydrodipicolinate synthase promoter-replaced strain The strain was constructed as described in 1. SMCT137 was used as the starting strain, pK18mobsacB-ΔPdapA::mPdapA was introduced into the starting strain, and the promoter of the 4-hydroxytetrahydrodipicolinate synthase gene was replaced. The resulting modified strain was named SMCT138, in which the promoter of the 4-hydroxytetrahydrodipicolinate synthase gene was replaced with a mutant dapA gene promoter (mPdapA).

[0072] 4. Construction of diaminopimelate dehydrogenase promoter replacement strain The strain was constructed as described in 1. SMCT137 was used as the starting strain, pK18mobsacB-ΔPddh::mPdapA was introduced into the starting strain, and the diaminopimelate dehydrogenase promoter was replaced. The resulting modified strain was named SMCT139, in which the promoter of the diaminopimelate dehydrogenase gene was replaced with a mutant dapA gene promoter (mPdapA). At the same time, pK18mobsacB-ΔPddh::mPdapA was introduced into SMCT138 as the starting strain, and modification was performed to replace the diaminopimelate dehydrogenase promoter. The obtained modified strain was named SMCT143.

[0073] 5. Construction of threonine dehydratase (ilvA) promoter replacement strain The strain was constructed as described in 1. Starting from SMCT137, pK18mobsacB-ΔPilvA::mPdapA was introduced into the starting strain to modify the threonine dehydratase (ilvA) promoter. The resulting modified strain was named SMCT140, in which the promoter of the threonine dehydratase gene ilvA was replaced with a mutant dapA gene promoter (mPdapA).

[0074] At the same time, pK18mobsacB-ΔPilvA::mPdapA was introduced into SMCT143 as the starting strain, and the threonine dehydratase (ilvA) promoter was replaced. The obtained modified strain was named SMCT144.

[0075] 6. Construction of threonine dehydratase (tdcB) promoter replacement strain The strain was constructed as described in 1. SMCT137 was used as the starting strain, pK18mobsacB-ΔPtdcB::mPdapA was introduced into the starting strain, and the threonine dehydratase (tdcB) promoter was replaced. The resulting modified strain was named SMCT141, in which the promoter of the threonine dehydratase gene tdcB was replaced with a mutant dapA gene promoter (mPdapA).

[0076] At the same time, pK18mobsacB-ΔPtdcB::mPdapA was introduced into the starting strain SMCT144 to modify the threonine dehydratase (tdcB) promoter replacement, and the obtained modified strain was named SMCT145.

[0077] 7. Construction of Citrate Synthase Promoter Replacement Strain The strain was constructed as described in 1. SMCT137 was used as the starting strain, pK18mobsacB-ΔPgltA::mPdapA was introduced into the starting strain, and the citrate synthase promoter was replaced. The resulting modified strain was named SMCT142, in which the citrate synthase gene promoter was replaced with a mutant dapA gene promoter (mPdapA).

[0078] At the same time, pK18mobsacB-ΔPgltA::mPdapA was introduced into the starting strain SMCT145 to modify the citrate synthase promoter, and the resulting modified strain was named SMCT146.

[0079] The constructed strains and their genotype information are shown in Table 2.

[0080] [Table 2]

[0081] Example 3 Shake flask fermentation validation of constructed strains Fermentation verification was carried out for the engineered fungus constructed in Example 2, and the specific method is as follows.

[0082] 1. Culture Medium Seed activation medium: BHI 3.7%, agar 2%, pH 7.0. Seed medium: peptone 5 / L, yeast extract 5g / L, sodium chloride 10g / L, ammonium sulfate 16g / L, urea 8g / L, potassium dihydrogen phosphate 10.4g / L, dipotassium hydrogen phosphate 21.4g / L, biotin 5mg / L, magnesium sulfate 3g / L, glucose 50g / L, pH 7.2. Fermentation medium: Corn steep liquor 50mL / L, glucose 30g / L, ammonium sulfate 4g / L, MOPS 30g / L, potassium dihydrogen phosphate 10g / L, urea 20g / L, biotin 10mg / L, magnesium sulfate 6g / L, ferrous sulfate 1g / L, VB1·HCl 40mg / L, calcium pantothenate 50mg / L, nicotinamide 40mg / L, manganese sulfate 1g / L, zinc sulfate 20mg / L, copper sulfate 20mg / L, pH 7.2.

[0083] 2. L-Threonine production by shake flask fermentation of engineered fungi (1) Seed culture: A loopful of slant culture seed of strains SMCT136, SMCT137, SMCT138, SMCT139, SMCT140, SMCT141, SMCT142, SMCT143, SMCT144, SMCT145, and SMCT146 was taken and inoculated into a 500 mL Erlenmeyer flask containing 20 mL of seed medium, and cultured with shaking at 30°C and 220 r / min for 16 h. (2) Fermentation culture: 2 mL of the seed solution was inoculated into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and cultured at 33°C and 220 r / min for 24 h with shaking. (3) 1 mL of the fermentation broth was taken and centrifuged (12,000 rpm, 2 min), the supernatant was collected, and the contents of L-threonine, lysine, and isoleucine in the fermentation broth of the engineered bacteria and the control bacteria were measured by HPLC.

[0084] Here, the fermentation results of the strains that have a rudimentary ability to synthesize threonine are shown in Table 3.

[0085] [Table 3]

[0086] As can be seen from the results in Table 3, in the strain in which aspartokinase was modified from the wild-type strain ATCC13032, threonine was initially accumulated, and with the enhancement of homoserine dehydrogenase expression, the threonine production was further improved, and 2.4 g / L of threonine could be accumulated.

[0087] The threonine synthesis status of the strains in which dapA, ddh, ilvA, tdcB and gltA were individually attenuated is shown in Table 4.

[0088] [Table 4]

[0089] As can be seen from the above results, the threonine production rates of the strains SMCT138, SMCT139, SMCT140, SMCT141, and SMCT142 were all improved to a certain extent compared to the starting strain SMCT137. By replacing the promoters of the lysine synthesis-related genes dapA and ddh with mPdapA, the lysine production rates were reduced by 0.3 and 0.2 g / L, respectively. By replacing the promoters of the isoleucine synthesis-related genes ilvA and tdcB with mPdapA, the isoleucine production rates were reduced by 0.6 and 0.2 g / L, respectively. By replacing the gltA promoter with mPdapA, the threonine production rate was increased by 0.8 g / L.

[0090] The fermentation results of the SMCT138, SMCT143, SMC144, SMCT145 and SMCT146 strains, in which five genes, dapA, ddh, ilvA, tdcB and gltA, were overlappingly attenuated, are shown in Table 5.

[0091] [Table 5]

[0092] As can be seen from the above results, after the carbon metabolic flow is stepwise optimized, the threonine production capacity is increased from 2.4g / L to 9.1g / L, which is an increase of 279%, which shows that the threonine production capacity of the strain can be significantly improved by optimizing the carbon metabolic flow after penetrating the threonine terminal synthesis pathway.

[0093] Although the present invention has been described in detail above using general descriptions and specific embodiments, it is clear to those skilled in the art that various modifications or improvements based on the present invention are possible. Therefore, any of these modifications or improvements made without departing from the spirit of the present invention are included in the scope of the claims of the present invention.

Claims

1. A promoter characterized by having the nucleotide sequence shown in SEQ ID NO.

1.

2. A biomaterial comprising the promoter of claim 1, which is a recombinant DNA, a vector or a host cell.

3. 10. Use of the promoter of claim 1 to initiate expression of a gene of interest.

4. Use of the promoter according to claim 1 for improving the production or conversion rate of a microbial metabolic product or for constructing a strain for producing the microbial metabolic product.

5. A recombinant microorganism, characterized in that a gene encoding at least one enzyme selected from the following (1) to (4) is transcriptionally driven by the promoter of claim 1: (1) 4-Hydroxytetrahydrodipicolinate synthase (2) Diaminopimelate dehydrogenase (3) Threonine dehydratase (4) Citrate synthase

6. In the recombinant microorganism, the enzyme activity of aspartokinase and / or homoserine dehydrogenase is enhanced and / or feedback inhibition is desensitized, The recombinant microorganism according to claim 5, wherein the enhancement of the enzyme activity is preferably achieved by the following 1) to 6), or any combination thereof: 1) Enhancement by introducing a plasmid carrying a gene encoding the enzyme 2) Enhancement by increasing the copy number of the gene encoding the enzyme in the chromosome 3) Enhancement by modifying the promoter sequence of the gene encoding the enzyme in the chromosome 4) Enhancement by operably linking a strong promoter to the gene encoding the enzyme 5) Enhancement by modifying the amino acid sequence of the enzyme 6) Enhancement by modifying the nucleotide sequence encoding the enzyme

7. 7. The recombinant microorganism according to claim 5, wherein the recombinant microorganism is a bacterium of the genus Corynebacterium, preferably Corynebacterium glutamicum.

8. a promoter of a gene encoding at least one of 4-hydroxytetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase, and citrate synthase is replaced with the promoter of claim 1; Preferably, the method further comprises enhancing the enzymatic activity of aspartokinase and / or homoserine dehydrogenase and / or relieving feedback inhibition, 7. The method for constructing a recombinant microorganism according to claim 5 or 6, wherein the enhancement of the enzyme activity is achieved by the following 1) to 6), or any combination thereof: 1) Enhancement by introducing a plasmid carrying a gene encoding the enzyme 2) Enhancement by increasing the copy number of the gene encoding the enzyme in the chromosome 3) Enhancement by modifying the promoter sequence of the gene encoding the enzyme in the chromosome 4) Enhancement by operably linking a strong promoter to the gene encoding the enzyme 5) Enhancement by modifying the amino acid sequence of the enzyme 6) Enhancement by modifying the nucleotide sequence encoding the enzyme

9. Any of the following uses: (1) Use for fermentative production of microbial metabolic products or their derivatives (2) Use for selecting strains that produce microbial metabolic products or their derivatives (3) Use for improving the production and / or conversion rate of microbial metabolic products 7. Use of a recombinant microorganism according to claim 5 or 6, wherein the microbial metabolic product is preferably threonine.

10. 7. A method for producing threonine or a derivative thereof by fermentation, comprising the steps of culturing the recombinant microorganism according to claim 5 or 6 and isolating and obtaining threonine or a derivative thereof from the culture.