Recombinant microorganisms producing threonine and uses thereof

Abstract

The present invention relates to the field of microbial engineering technology, specifically to a recombinant microorganism producing threonine and its use. The present invention provides a recombinant microorganism in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter. The present invention significantly improves the threonine synthesis ability of a strain by specific optimization of the promoter of a phosphoenolpyruvate carboxylase-encoding gene and mutation of the gene coding region, and the threonine production is increased by 25-40% compared with a non-modified strain. The modification method can be used for constructing a threonine-producing strain and fermentative production of threonine, and has good use value.

Description

[Technical field]

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

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

[0003] In Corynebacterium glutamicum, the production of threonine from oxaloacetate requires five catalytic steps, each of which is catalyzed by the enzymes 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).Currently, the production of threonine using Corynebacterium glutamicum has been reported mainly by modifying the metabolic pathway of threonine synthesis. Among these, the production of homoserine dehydrogenase and aspartokinase resistant to feedback inhibition was achieved (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.); and attenuating the coding gene glyA in the threonine utilization pathway and overexpressing the threonine exporter 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.), but there are still few reports on the supply of precursors for threonine production. Summary of the Invention

[0004] An object of the present invention is to provide a threonine-producing recombinant microorganism having improved threonine-producing ability by modifying the promoter region of a gene encoding phosphoenolpyruvate carboxylase, and use thereof.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] First, the present invention provides use of enhanced expression of a phosphoenolpyruvate carboxylase-encoding gene (ppc) for improving threonine production in a Corynebacterium bacterium or for constructing a Corynebacterium bacterium that produces threonine, wherein the enhanced expression is achieved by replacing 20 to 30 bp upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene with a strong promoter.

[0007] The bacterium of the genus Corynebacterium is preferably Corynebacterium glutamicum.

[0008] The present invention has discovered that improving the enzyme activity of phosphoenolpyruvate carboxylase in Corynebacterium glutamicum is advantageous for improving the supply of oxaloacetate, which is a synthetic precursor of threonine, and further promotes the synthesis and accumulation of threonine. As a result of many attempts and screening for means for improving the enzyme activity of phosphoenolpyruvate carboxylase, the present invention has unexpectedly discovered that the expression level of phosphoenolpyruvate carboxylase can be significantly improved by replacing a specific region in the upstream non-coding region of the coding gene with a strong promoter. Such a modification is more stable than increasing the copy number of the gene, and the effect of improving the enzyme activity of phosphoenolpyruvate carboxylase by this modification is obviously superior to that of replacing the ppc promoter with a stronger promoter.

[0009] Preferably, said enhanced expression is due to replacement of the 27 bp upstream of the start codon of the phosphoenolpyruvate carboxylase coding gene with a strong promoter.

[0010] More preferably, the strong promoter is Ptuf. The nucleotide sequence of the strong promoter Ptuf is shown in SEQ ID NO.4.

[0011] In order to enhance gene expression, the original promoter of the gene is usually replaced with a strong promoter, or a strong promoter is directly inserted upstream of the start codon of the gene. When replacing the promoter, the spacer sequence between the target gene and the upstream gene is usually replaced with a strong promoter, or the original promoter region of the target gene is replaced with a strong promoter. However, the present invention has found that the effect of improving the expression level of phosphoenolpyruvate carboxylase is obviously superior to that of replacing the promoter region of the phosphoenolpyruvate carboxylase-encoding gene by replacing the 27 bp DNA fragment upstream of the start codon with a strong promoter (especially the strong promoter Ptuf).

[0012] The 27-bp fragment upstream of the start codon in the present invention refers to a DNA fragment having a length of 27 bp obtained by extending upstream from the first base upstream of the start codon to the 27th base. Taking the wild-type Corynebacterium glutamicum strain ATCC13032 as an example, the sequence of the 27-bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene is shown in SEQ ID NO.3.

[0013] The amino acid sequence of the phosphoenolpyruvate carboxylase according to the present invention is that shown in SEQ ID NO. 1 or 2, or is an amino acid sequence having at least 90% identity to the sequence shown in SEQ ID NO. 1 or 2 and having an equivalent function.

[0014] Preferably, the amino acid sequence of the phosphoenolpyruvate carboxylase is shown in SEQ ID NO. 1 or 2.

[0015] Here, the sequence shown in SEQ ID NO.1 is the amino acid sequence of wild-type phosphoenolpyruvate carboxylase of Corynebacterium glutamicum. The sequence shown in SEQ ID NO.2 is a mutant protein (D299N) in which D at position 299 in wild-type phosphoenolpyruvate carboxylase of Corynebacterium glutamicum is mutated to N. The threonine production of the strain can be significantly improved by combining the D299N mutation of phosphoenolpyruvate carboxylase with the modification of the promoter region. The amino acid sequence of phosphoenolpyruvate carboxylase is preferably shown in SEQ ID NO.2.

[0016] Furthermore, the present invention provides a recombinant microorganism in which a 20-30 bp fragment upstream of the initiation codon of a phosphoenolpyruvate carboxylase-encoding gene has been replaced with a strong promoter.

[0017] Preferably, in said recombinant microorganism, the 27 bp fragment upstream of the initiation codon of the phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter.

[0018] More preferably, the strong promoter is Ptuf.

[0019] The amino acid sequence of the phosphoenolpyruvate carboxylase is shown in SEQ ID NO. 1 or 2, or an amino acid sequence having at least 90% identity to the sequence shown in SEQ ID NO. 1 or 2 and having an equivalent function.

[0020] The amino acid sequence of phosphoenolpyruvate carboxylase is preferably as shown in SEQ ID NO.1 or 2. In addition to the modification of the promoter region, the D299N mutation of phosphoenolpyruvate carboxylase can be combined to significantly improve the threonine production of the strain. Therefore, the amino acid sequence of phosphoenolpyruvate carboxylase of the recombinant microorganism is preferably as shown in SEQ ID NO.2.

[0021] The recombinant microorganism as described above is preferably constructed by a method of replacing a 27 bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene of the starting strain with the strong promoter Ptuf, and preferably further comprising a step of mutating the phosphoenolpyruvate carboxylase-encoding gene to generate a D299N mutation in the phosphoenolpyruvate carboxylase.

[0022] Here, the starting strain is preferably a strain capable of accumulating threonine.

[0023] Preferably, in the recombinant microorganism, any one or more of the following enzymes (1) to (7) have enhanced enzyme activity and / or feedback inhibition is relieved: (1) Aspartokinase (2) Aspartate semialdehyde dehydrogenase (3) Homoserine dehydrogenase (4) Threonine synthase (5) Homoserine kinase (6) Aspartate aminotransferase (7) Threonine exporter

[0024] Preferably, the threonine exporter is a threonine exporter derived from Escherichia coli.

[0025] The aspartokinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotreatase, homoserine kinase, and threonine synthase have NCBI reference sequence numbers WP_003855724.1, WP_003854900.1, WP_011013506.1, WP_011013497.1, WP_011014183.1, and WP_011014964.1, respectively, or are amino acid sequences having at least 90% identity to the reference sequences and equivalent functions.

[0026] The Escherichia coli threonine exporter has the reference sequence YP_026264.1 on NCBI or an amino acid sequence having at least 90% identity to the reference sequence and having an equivalent function.

[0027] Preferably, the microorganism is one of the following: 1 )~( 6 ) ( 1 ) a microorganism in which a 20-30 bp fragment upstream of the initiation codon of a phosphoenolpyruvate carboxylase-encoding gene has been replaced with a strong promoter, and in which the activity of aspartate aminotransferase has been enhanced and / or feedback inhibition has been relieved; ( 2 ) a microorganism in which a 20-30 bp fragment upstream of the initiation codon of a phosphoenolpyruvate carboxylase-encoding gene has been replaced with a strong promoter, and the activities of aspartate aminotransferase, aspartokinase and / or aspartate semialdehyde dehydrogenase have been enhanced and / or feedback inhibition has been relieved; ( 3 ) a microorganism in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene has been replaced with a strong promoter, and the activities of aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase and / or homoserine dehydrogenase have been enhanced and / or feedback inhibition has been relieved; ( 4 ) a microorganism in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene has been replaced with a strong promoter, and in which the activities of aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase and / or homoserine kinase have been enhanced and / or feedback inhibition has been relieved; ( 5 ) a microorganism in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene has been replaced with a strong promoter, and in which the activities of aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and / or threonine synthase have been enhanced and / or feedback inhibition has been relieved; ( 6 ) A microorganism in which a 20 to 30 bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene has been replaced with a strong promoter, and the activities of aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine synthase and / or threonine exporter have been enhanced and / or feedback inhibition has been relieved.

[0028] The enhancement of the enzyme activity can be 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

[0029] In a preferred embodiment of the present invention, the recombinant microorganism is any one of the following (1) to (6). (1) The D299N mutation was introduced into phosphoenolpyruvate carboxylase, and the 27-bp fragment upstream of the start codon of the coding gene was replaced with the Ptuf promoter, resulting in enhanced expression of aspartate aminotransferase. (2) The D299N mutation site was introduced into phosphoenolpyruvate carboxylase, and the 27-bp fragment upstream of the start codon of the coding gene was replaced with the Ptuf promoter. At the same time, aspartate aminotransferase, aspartokinase, and aspartate semialdehyde dehydrogenase were enhanced in expression, and the feedback inhibition of aspartase was released. (3) The D299N mutation site was introduced into phosphoenolpyruvate carboxylase, and the 27-bp fragment upstream of the start codon of the coding gene was replaced with the Ptuf promoter. At the same time, aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, and homoserine dehydrogenase were enhanced in expression, and the feedback inhibition of aspartokinase and homoserine dehydrogenase was released. (4) A mutation site D299N was introduced into phosphoenolpyruvate carboxylase, and a 27-bp fragment upstream of the start codon of the coding gene was replaced with the Ptuf promoter. At the same time, aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, and homoserine kinase were enhanced in expression, and the feedback inhibition of aspartokinase and homoserine dehydrogenase was released. (5) A mutation site D299N was introduced into phosphoenolpyruvate carboxylase, and a 27-bp fragment upstream of the start codon of the coding gene was replaced with the Ptuf promoter. At the same time, aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase were enhanced in expression, and the feedback inhibition of aspartokinase and homoserine dehydrogenase was released. (6) The mutation site D299N was introduced into phosphoenolpyruvate carboxylase, and the 27 bp upstream of the ATG start codon of the coding gene was replaced with the Ptuf promoter. At the same time, aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase were enhanced in expression, and the threonine exporter derived from Escherichia coli was expressed, and the feedback inhibition of aspartokinase and homoserine dehydrogenase was released.

[0030] Preferably, the enhancement or enhanced expression of the enzyme activity as described above is achieved by replacing the original promoter of the gene with a strong promoter, or by mutating the start codon of the gene so that the first amino acid of the protein encoded thereby is mutated from valine to methionine.

[0031] Preferably, the strong promoter is the promoter Psod, PcspB or Ptuf, the nucleotide sequences of which are shown in SEQ ID NOs. 5 and 6, respectively.

[0032] Preferably, enhanced expression of the genes encoding aspartokinase and threonine synthase is achieved by replacing their original promoters with the promoter Psod and by replacing their initiation codons GTG by ATG.

[0033] Enhanced expression of the genes encoding aspartate semialdehyde dehydrogenase and aspartate aminotransferase is achieved by replacing their original promoters with the promoter Psod.

[0034] Enhanced expression of the gene encoding homoserine dehydrogenase and the gene encoding homoserine kinase is achieved by replacing their original promoters with the promoter PcspB.

[0035] The expression of the E. coli threonine exporter can be achieved by integrating an expression cassette of a gene encoding the E. coli threonine exporter into the chromosome of the microorganism. The preferred integration site is downstream of the cg2009 gene, and the promoter of the expression cassette is Psod.

[0036] The feedback resistant aspartokinase is preferably obtained by a T311I mutation.The feedback resistant homoserine dehydrogenase is preferably obtained by a G378E mutation. The microorganism used to construct the recombinant microorganism as described above 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.

[0037] The present invention also provides a method for constructing said recombinant microorganism, which comprises replacing a 27 bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene of the starting strain with the strong promoter Ptuf.

[0038] Preferably, the method further comprises mutating the phosphoenolpyruvate carboxylase-encoding gene to produce a D299N mutation in the phosphoenolpyruvate carboxylase encoded thereby.

[0039] More preferably, the method further comprises enhancing the enzymatic activity of any one or more of the following enzymes (1) to (7) and / or releasing the feedback inhibition thereof: (1) Aspartokinase, (2) aspartate semialdehyde dehydrogenase, (3) homoserine dehydrogenase, (4) threonine synthase, (5) homoserine kinase, (6) aspartate aminotransferase, (7) threonine exporter,

[0040] 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

[0041] The above modification methods, including gene enhancement, are all known to those skilled in the art, and can be referred to as follows: 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 Bacterial Strain Producing L-Isoleucine and Optimization of Fermentation Conditions, Tianjin University of Science and Technology, 2015.

[0042] The present invention provides any one of the following uses of the recombinant microorganism: (1) Use in fermentation production of threonine or its derivatives (2) Use as a starting strain for constructing a strain producing threonine or its derivatives (3) Use for improving the production amount and / or yield of threonine or its derivatives

[0043] 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.

[0044] Specifically, the method includes inoculating the recombinant microorganism into a seed medium to obtain a seed liquor, inoculating the seed liquor into a fermentation medium to obtain a fermentation liquor, and separating and extracting the fermentation liquor to obtain threonine or a derivative thereof.

[0045] Preferably, the fermentation medium contains the following components: corn steep liquor 45-55 mL / L, glucose 25-35 g / L, ammonium sulfate 3-5 g / L, MOPS 25-35 g / L, potassium dihydrogen phosphate 8-12 g / L, urea 15-25 g / L, biotin 8-12 mg / L, magnesium sulfate 5-7 g / L, ferrous sulfate 0.5-1.5 g / L, VB1·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.

[0046] According to the present invention, by specifically optimizing the promoter of the phosphoenolpyruvate carboxylase-encoding gene and mutating the gene coding region, the threonine synthesis ability of the strain is significantly improved, and the threonine production amount is increased by 25-40% compared with that of the unmodified strain, which has the beneficial effect. The modification method can be used for constructing a threonine-producing strain and fermenting and producing threonine, and has good use value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The following examples are intended to illustrate the present invention and are not intended to limit the scope of the invention.

[0048] The present invention focuses on the effect of modifying the promoter region of phosphoenolpyruvate carboxylase on threonine production, and has confirmed that threonine production can be significantly promoted by replacing 20 to 30 bp, preferably 27 bp, upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene with a strong promoter.

[0049] The present invention further enhances the threonine synthesis and transport pathway of the strain, mainly including enhanced or deregulated expression of aspartokinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase, threonine synthase, and threonine exporter. The shake flask results show that all threonine-producing strains have improved threonine production ability after replacing the upstream 20-30bp of the start codon of their phosphoenolpyruvate carboxylase-encoding gene with a strong promoter, and also show that the strains that combine replacing the upstream 20-30bp of the start codon of the phosphoenolpyruvate carboxylase-encoding gene with a strong promoter and enhancing the expression of enzymes in the threonine synthesis / transport pathway are more advantageous in threonine production than the strains that only replace the upstream 20-30bp of the start codon of the phosphoenolpyruvate carboxylase-encoding gene with a strong promoter.

[0050] The expression enhancement in the modification process includes promoter replacement, ribosome binding site modification, copy number increase, plasmid overexpression, etc., all of which are known to those skilled in the art. The above means are not exhaustive, and in the specific examples, only promoter enhancement will be described as a representative example.

[0051] The present invention adopts the following technical solutions:

[0052] Technical Scheme 1 of the present invention provides a method for producing threonine using a strain in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter, and aspartate aminotransferase expression is enhanced and / or deregulated.

[0053] Technical solution 2 of the present invention provides a method for producing threonine using a strain in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter, and at least one of aspartate aminotransferase, aspartokinase, and aspartate semialdehyde dehydrogenase is expressed enhanced and / or deregulated.

[0054] Technical Scheme 3 of the present invention provides a method for producing threonine using a strain in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter, and at least one of aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, and homoserine dehydrogenase is expressed enhanced and / or deregulated.

[0055] Technical solution 4 of the present invention provides a method for producing threonine using a strain in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter, and at least one of aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, and homoserine kinase is enhanced and / or deregulated.

[0056] Technical solution 5 of the present invention provides a method for producing threonine using a strain in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter, and at least one of aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase is expressed enhanced and / or deregulated.

[0057] Technical solution 6 of the present invention provides a method for producing threonine using a strain in which a 20-30 bp fragment upstream of the start codon of a phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter, and at least one of aspartate aminotransferase, aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine synthase, and threonine exporter is enhanced and / or deregulated.

[0058] The strain is a bacterium of the genus Corynebacterium, preferably Corynebacterium glutamicum, and most preferably Corynebacterium glutamicum ATCC 13032.

[0059] Details of the enzyme and gene according to the present invention are as follows. Aspartokinase: coding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247, Aspartate semialdehyde dehydrogenase: coding gene name asd, NCBI number: Cgl0252, Cg0307, ​​NCgl0248, Homoserine dehydrogenase: coding gene name hom, NCBI number: Cg1337, Cgl1183, NCgl1136, Threonine synthase: coding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139, Homoserine kinase: coding gene thrB, NCBI numbers: Cgl1184, Cg1338, NCgl1137, Phosphoenolpyruvate carboxylase: coding gene ppc, NCBI numbers: cg1787, Cgl1585, NCgl1523, Aspartate aminotransferase: coding gene name aspB, NCBI number: cg0294, cg0294 and cg0294, Threonine exporter from Escherichia coli: coding gene name rhtC, NCBI number: 948317. EXAMPLES

[0060] Example 1 Construction of a plasmid for modifying the genome of a strain

[0061] 1. Aspartokinase-aspartate semialdehyde dehydrogenase operon expression enhancing plasmid pK18mobsacB-P sod -lysC g1a-T311I -Building asd 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 fragment up-Psod was obtained by fusion PCR using the P21 / P24 primer set with up and Psod as templates. 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 to obtain up-Psod-lysC. g1a-T311I The -dn was assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I -asd was obtained.

[0062] 2. Aspartate Aminotransferase Expression Enhancement Plasmid pK18mobsacB-P sod -Construction of aspB The plasmid construction method was as described above in 1, and the primers used were P103, P104, P105, P106, P107 and P108.

[0063] 3. Homoserine dehydrogenase-homoserine kinase operon expression enhancing plasmid pK18mobsacB-PcspB -hom G378E Construction of -thrB The plasmid construction method was as described above in 1, and the primers used were P29, P30, P31, P32, P33, P34, P35, and P36.

[0064] 4. Threonine synthase expression-enhancing plasmid pK18mobsacB-P sod -thrC g1a Construction The plasmid construction method was as described above in 1, and the primers used were P37, P38, P39, P40, P41 and P42.

[0065] 5. Threonine exporter expression enhancing plasmid pK18mobsacB-P sod -Building rhtC The plasmid construction method was as described above in 1, and the primers used were P157, P158, P159, P160, P161, P162, P163 and P164.

[0066] 6. Phosphoenolpyruvate carboxylase expression enhancing plasmid pK18mobsacB-Ptuf-ppc D299N Construction Using the ATCC13032 genome as a template, PCR amplification was performed with the P53 / P54 primer set to obtain the upstream homology arm up, with the P55 / P56 primer set to obtain the promoter fragment Ptuf, and with the P57 / P58 primer set to obtain ppc D299N The downstream homology arm dn was obtained by PCR amplification using the P59 / P60 primer set. The fragment up-Ptuf was obtained by fusion PCR using the P53 / P56 primer set with up and Ptuf as templates. D299N Fusion PCR was performed using dn as a template and the P53 / P60 primer set to generate the full-length fragment up-Ptuf-ppc D299N pK18mobsacB was digested with BamHI / HindIII to obtain up-Ptuf-ppc. D299NThe -dn was assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-Ptuf-ppc D299N obtained.

[0067] The primers used in the above plasmid construction process are shown in Table 1.

[0068] [Table 1]

[0069] Example 2 Construction of genome modified strain

[0070] 1. Construction of Aspartate Aminotransferase Enhanced Expression Strain ATCC13032 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-P sod The competent cells were transformed with -aspB by electroporation, and transformants in which the target gene had been integrated into the chromosome by homology were selected on a selection 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 on 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 resulting strain was serially diluted to 1000 μg / ml, and the dilutions were spread onto standard solid brain heart infusion medium containing 10% sucrose and incubated at 33°C for 48 h. The genome of the colonies grown on the sucrose medium did not carry the integrated vector sequence. The target fragment was amplified by PCR and subjected to nucleotide sequencing analysis, and the obtained target mutant strain was named SMCT061. In this strain, the promoter of the aspB gene was replaced with the Psod promoter compared to the ATCC13032 strain.

[0071] 2. Construction of Aspartokinase-Aspartate Semialdehyde Dehydrogenase Operon Enhanced Expression Strain The strain construction method was as described above in 1. Starting from SMCT061, pK18mobsacB-P sod -lysC g1a-T311I The -asd plasmid was introduced into SMCT061 to enhance the aspartokinase-aspartate semialdehyde dehydrogenase operon, and the resulting strain was named SMCT062. Compared to the SMCT061 strain, this strain has a mutation in the lysC gene, in which the initiation codon has been mutated from GTG to ATG, the 311th amino acid in the encoded sequence has been mutated from threonine to isoleucine, and the promoter of the lysC-asd operon has been replaced with the Psod promoter.

[0072] 3. Construction of homoserine dehydrogenase-homoserine kinase expression-enhanced strain The strain construction method was as described above in 1. Starting from SMCT062, pK18mobsacB-P cspB -hom G378E The -thrB plasmid was introduced into SMCT062 to enhance homoserine dehydrogenase-homoserine kinase expression, and the resulting modified strain was named SMCT063. Compared to the SMCT062 strain, this strain has a G378E mutation in the encoded protein due to a mutation in the hom gene, and the promoter of the hom-thrB operon is P cspB It has been replaced by a promoter.

[0073] 4. Construction of Threonine Synthase Expression Enhanced Strain The strain construction method was as described above in 1. Starting from SMCT063, pK18mobsacB-P sod -thrC g1a The plasmid was introduced into SMCT063 to enhance threonine synthase expression, and the resulting modified strain was named SMCT064. Compared to the SMCT063 strain, this strain has a mutated start codon of the thrC gene to ATG and a promoter of the thrC gene to P sod has been replaced by.

[0074] 5. Construction of Threonine Exporter Expression Enhanced Strain The strain construction method was as described above in 1. Starting from SMCT064, pK18mobsacB-P sod The -rhtC plasmid was introduced into SMCT064 to enhance threonine exporter expression, and the resulting modified strain was named SMCT065. This strain has the rhtC gene, a threonine exporter gene derived from E. coli, inserted downstream of the cg2009 gene, in comparison with SMCT064.

[0075] 6. Construction of phosphoenolpyruvate carboxylase expression-enhanced strain The strain construction method was as described above in 1. Starting strains were SMCT061, SMCT062, SMCT063, SMCT064, and SMCT065, and pK18mobsacB-Ptuf-ppc D299N The plasmids were introduced into the starting strains to enhance phosphoenolpyruvate carboxylase expression, and the resulting modified strains were named SMCT066, SMCT067, SMCT068, SMCT079, and SMCT070. Compared to the corresponding starting strains, these modified strains have a D299N mutation in the encoded protein due to a mutation in the phosphoenolpyruvate carboxylase gene ppc, and the 27-bp fragment upstream of the start codon of the ppc gene has been replaced with the Ptuf promoter.

[0076] The genotypes of the strains obtained by the above genetic modifications are shown in Table 2.

[0077] [Table 2]

[0078] Example 3 Shake flask fermentation validation of strains Shake flask fermentation verification was carried out for each of the modified strains constructed in Example 2, specifically as follows.

[0079] 1. Culture Medium Seed activation medium: BHI 3.7%, agar 2%, pH 7. 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.

[0080] 2. L-Threonine production by shake flask fermentation of engineered fungi (1) Seed culture: A loopful of slant culture seed of SMCT061, SMCT062, SMCT063, SMCT064, SMCT065, SMCT066, SMCT067, SMCT068, SMCT069 and SMCT070 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 hours to obtain a seed solution. (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 hours with shaking to obtain a fermentation liquid. (3) 1 mL of the fermentation liquid was taken and centrifuged (12,000 rpm, 2 min) to recover the supernatant, and L-threonine in the fermentation liquid of the engineered bacteria and the control bacteria was measured by HPLC.

[0081] The shake flask fermentation results for threonine are shown in Table 3.

[0082] [Table 3]

[0083] As can be seen from the results, in the threonine-producing strains SMCT061, SMCT062, SMCT063, SMCT064, and SMCT065, the promoter of the phosphoenolpyruvate carboxylase coding gene is optimized, and the phosphoenolpyruvate carboxylase is engineered to undergo D299N mutation, thereby further improving the production of threonine, and the threonine production of the strains SMCT066, SMCT067, SMCT068, SMCT069, and SMCT070 is improved by 25%, 33%, 35%, 38%, and 40%, respectively. By replacing the 20-30 bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase coding gene with a strong promoter, and by engineering phosphoenolpyruvate carboxylase to undergo D299N mutation, the supply of oxaloacetate, which is a synthetic precursor of threonine, can be increased, and the synthesis of threonine can be further promoted. In addition, the production of threonine is further improved by combining the modification of the phosphoenolpyruvate carboxylase with the enhanced expression of different enzymes in the threonine synthesis pathway and the threonine exporter, which shows that the combination of the above modifications is more advantageous in improving the threonine production ability of the strain.

[0084] 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 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. 2. Use of enhanced expression of a phosphoenolpyruvate carboxylase-encoding gene for improving threonine production in a Corynebacterium bacterium or constructing a Corynebacterium bacterium that produces threonine, comprising: The use of enhanced expression of a phosphoenolpyruvate carboxylase-encoding gene, characterized in that the enhanced expression is achieved by replacing a 20-30 bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene with a strong promoter.

2. The enhanced expression is achieved by replacing a 27 bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene with a strong promoter; 2. Use according to claim 1, characterized in that preferably said strong promoter is Ptuf.

3. The use according to claim 1 or 2, characterized in that the amino acid sequence of the phosphoenolpyruvate carboxylase is shown in SEQ ID NO. 1 or 2.

4. A 20-30 bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase coding gene was replaced with a strong promoter. Preferably, the 27 bp fragment upstream of the start codon of the phosphoenolpyruvate carboxylase-encoding gene is replaced with a strong promoter; More preferably, the recombinant microorganism is characterized in that the strong promoter is Ptuf.

5. The recombinant microorganism according to claim 4, characterized in that the amino acid sequence of the phosphoenolpyruvate carboxylase of the recombinant microorganism is shown in SEQ ID NO. 1 or 2.

6. The recombinant microorganism according to claim 4 or 5, characterized in that in the recombinant microorganism, the enzymatic activity of any one or more of the following enzymes (1) to (7) is enhanced and / or feedback inhibition is relieved: (1) Aspartokinase (2) Aspartate semialdehyde dehydrogenase (3) Homoserine dehydrogenase (4) Threonine synthase (5) Homoserine kinase (6) Aspartate aminotransferase (7) A threonine exporter, preferably a threonine exporter derived from Escherichia coli.

7. The recombinant microorganism according to claim 6, characterized in that 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

8. The recombinant microorganism according to any one of claims 4 to 7, characterized in that the microorganism used to construct the recombinant microorganism is a bacterium of the genus Corynebacterium, preferably Corynebacterium glutamicum.

9. Any of the following uses of a recombinant microorganism according to any one of claims 4 to 8: (1) Use in fermentation production of threonine or its derivatives (2) Use as a starting strain for engineering a strain producing threonine or its derivatives. (3) Use for improving the production amount and / or yield of threonine or its derivatives

10. A method for producing threonine or a derivative thereof by fermentation, comprising the steps of culturing the recombinant microorganism according to any one of claims 4 to 8 and isolating and obtaining threonine or a derivative thereof from the culture.

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