Methods for constructing engineered bacteria that produce threonine

Abstract

The present invention provides a method for constructing a threonine-producing engineered strain. The present invention uses a 2-methyl-citrate synthase 1-inactivated strain (Corynebacterium) for threonine production, which can increase the amount of threonine produced by about 42% compared to an unmodified strain. Furthermore, when combined with enhanced expression of at least one of aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase, fructose-1,6-diphosphatase 2, etc. in the threonine synthesis pathway, the amount of threonine produced can all be improved. The present invention provides a new approach to large-scale threonine production and has high utility value.

Description

[Technical field]

[0001] The present invention belongs to the field of microbial engineering technology, and specifically relates to a method for constructing an engineered fungus capable of producing threonine. [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, pharmaceuticals, food supplements, and feed additives.

[0003] In Corynebacterium glutamicum, the production of threonine from oxaloacetate requires five catalytic steps catalyzed by 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), respectively. Hermann Sahm and his colleagues have been working on the development of a highly threonine-producing Corynebacterium glutamicum and have made some breakthroughs, such as obtaining feedback-resistant homoserine dehydrogenase (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) and aspartokinase (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).Lothar Eggling, following Hermann Sahm, further explored this field and increased threonine production from 49mM to 67mM by attenuating 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 using Corynebacterium glutamicum have focused mainly on its synthetic pathway, with few reports on precursor supply, etc. Furthermore, previous reports are merely initial studies on the synthetic pathway of threonine, and have not yet been systematized. Summary of the Invention

[0005] The object of the present invention is to provide a method for constructing a threonine (L-threonine)-producing engineered strain by inactivating 2-methyl-citrate synthase 1, thereby improving the threonine-producing ability of the strain.

[0006] In order to achieve the object of the present invention, in a first aspect, the present invention provides a modified Corynebacterium microorganism having a reduced or lost activity of 2-methyl-citrate synthase 1 compared to an unmodified microorganism, and having an enhanced threonine production ability compared to an unmodified microorganism. Preferably, the 2-methyl-citrate synthase 1 is an amino acid sequence having the reference sequence number WP_011013823.1 on NCBI or 90% identity thereto.

[0007] Furthermore, the reduction or loss of activity of 2-methyl-citrate synthase 1 in the microorganism is achieved by reducing the expression of the gene encoding 2-methyl-citrate synthase 1 or by knocking out the gene encoding endogenous 2-methyl-citrate synthase 1.

[0008] Methods such as mutagenesis, site-directed mutagenesis, or homologous recombination can be used to reduce expression of the gene encoding 2-methyl-citrate synthase 1 or to knock out endogenous 2-methyl-citrate synthase 1-encoding genes.

[0009] Furthermore, the microorganism has enhanced activity of an enzyme involved in the threonine synthesis pathway in its body, as compared to an unmodified microorganism.

[0010] Here, the enzyme related to the threonine synthesis pathway is selected from at least one of aspartate aminothreonase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase, and fructose-1,6-diphosphatase 2. Preferably, the reference sequence numbers on NCBI for these are WP_011013497.1, WP_003855724.1, WP_003854900.1, WP_011014964.1, NP_600631.1, and WP_003856830.1, respectively, or an amino acid sequence having 90% identity to the reference sequence.

[0011] Preferably, the microorganism is one of the following: 1 )~( 5 ) ( 1 ) A microorganism in which the activity of 2-methyl-citrate synthase 1 is reduced or absent and the activity of aspartokinase and / or homoserine dehydrogenase is enhanced. ( 2) A microorganism in which the activity of 2-methyl-citrate synthase 1 is reduced or absent and the activities of aspartokinase, homoserine dehydrogenase and / or threonine synthase are enhanced. ( 3 ) A microorganism in which the activity of 2-methyl-citrate synthase 1 is reduced or absent and the activities of aspartokinase, homoserine dehydrogenase and / or NAD kinase are enhanced. ( 4 ) A microorganism in which the activity of 2-methyl-citrate synthase 1 is reduced or absent, and the activity of aspartokinase, homoserine dehydrogenase, NAD kinase and / or fructose-1,6-diphosphatase 2 is enhanced. ( 5 ) A microorganism in which the activity of 2-methyl-citrate synthase 1 is decreased or absent, and the activities of aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase, and fructose-1,6-diphosphatase 2 are increased.

[0012] The enhancement of the activity of an enzyme involved in the threonine synthesis pathway in the microbial body 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

[0013] Preferably, the Corynebacterium microorganism used in the present invention is Corynebacterium glutamicum, which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum phylogenetic tree https: / / www.ncbi.nlm.nih.gov / genome / 469), with Corynebacterium glutamicum ATCC 13032 being more preferred.

[0014] In a second aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: A. Attenuating the gene encoding 2-methyl-citrate synthase 1 in an amino acid producing Corynebacterium to obtain a gene-attenuated strain, and / or B. enhancing an enzyme involved in the threonine synthesis pathway in the gene-weakened strain obtained in step A to obtain a strain with enhanced enzyme activity; The attenuation comprises knocking out or reducing the expression of a gene encoding 2-methyl-citrate synthase 1; The present invention provides a method for constructing a threonine-producing engineered strain, wherein the enhancement is selected from the following 1) to 5) 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

[0015] In a third aspect, the present invention provides a method for producing a composition comprising the steps of: a) culturing the modified Corynebacterium microorganism to obtain a culture of the microorganism; b) recovering threonine from the culture obtained in step a).

[0016] In a fourth aspect, the present invention provides the use of knockout or down-regulation of the gene encoding 2-methyl-citrate synthase 1 for the fermentative production of threonine or for improving the fermentative production yield of threonine.

[0017] Furthermore, the fermentation production of threonine is improved by inactivating 2-methyl-citrate synthase 1 in Corynebacterium having amino acid production ability.

[0018] Preferably, the Corynebacterium used in the present invention is Corynebacterium glutamicum, which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum phylogenetic tree https: / / www.ncbi.nlm.nih.gov / genome / 469), with Corynebacterium glutamicum ATCC 13032 being more preferred.

[0019] According to a fifth aspect, the present invention provides use of the modified Corynebacterium microorganism or the engineered threonine-producing strain constructed by the method for fermentative production of threonine or for improving the fermentative production amount of threonine.

[0020] The modification methods for the above strains, including gene strengthening and weakening, 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.

[0021] According to the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0022] The present invention inactivates 2-methyl-citrate synthase 1 to enhance the supply of oxaloacetate, which is a synthetic precursor of threonine, and improves the threonine production ability of the strain. The present invention uses a 2-methyl-citrate synthase 1 inactivated strain (Corynebacterium, for example, Corynebacterium glutamicum) for threonine production, thereby improving the production amount of threonine by about 60% compared to a non-modified strain. Furthermore, when it is combined with the enhanced expression of at least one of aspartate aminothreonase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase, fructose-1,6-diphosphatase 2, etc. in the threonine synthesis pathway, the production amount of threonine can be improved. The present invention provides a new approach to the large-scale production of threonine and has high utility value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention improves threonine production in a bacterial strain (eg, Corynebacterium glutamicum) by inactivating 2-methyl-citrate synthase 1 (prpC1).

[0024] 2-Methyl-citrate synthase 1 is not directly related to the threonine synthesis pathway, and there is currently no relevant report that the downstream product of threonine can be increased by inactivating 2-methyl-citrate synthase 1. Therefore, in the present invention, a 2-methyl-citrate synthase 1-inactivated strain was constructed using Corynebacterium glutamicum ATCC 13032 as a starting strain, but the threonine production of the obtained bacterium was unexpectedly low at 0.2 g / L, and it was presumed that in the threonine synthesis, aspartokinase and homoserine dehydrogenase in the threonine synthesis pathway are strictly controlled by the intracellular threonine concentration. Therefore, in order to produce threonine by modifying the strain, the synthetic pathway is first penetrated by obtaining and enhancing the expression of aspartokinase and homoserine dehydrogenase, which are resistant to feedback inhibition, to obtain the modified strain SMCT077 with the initial threonine synthesis ability, and the threonine production amount is 2.4g / L. By further inactivating prpC1, the threonine production amount is improved to 3.3g / L. From this, it was found that the inactivation of 2-methyl-citrate synthase 1 is advantageous for the production of threonine, but when it is combined with other sites related to threonine synthesis in the threonine-producing strain, the threonine production ability is further improved.

[0025] To further verify that the improvement in threonine production was due to the inactivation of 2-methyl-citrate synthase 1, a series of strains was obtained by inactivating prpC1 in SMCT077 strains in which at least one of the enzymes aspartate aminotransferase, threonine synthase, NAD kinase, and fructose-1,6-bisphosphatase was further enhanced in expression. All of the prpC1-inactivated strains showed a 42% improvement in threonine production.

[0026] The expression enhancement in the modification process includes promoter replacement, ribosome binding site modification, copy number increase, activity increase by amino acid sequence modification, and plasmid overexpression, while the inactivation includes expression activity reduction and activity loss, all of which are known to those skilled in the art. The above means are not shown comprehensively by examples, and in the specific examples, only promoter enhancement is described as a representative. In addition, the present invention shows only some combinations of modifications, but any combination of the above sites can improve the production of threonine, and the present text is merely an illustrative description and does not comprehensively describe everything.

[0027] The present invention adopts the following technical solutions:

[0028] Technical Scheme 1 of the present invention provides a method for producing threonine using Corynebacterium in which 2-methyl-citrate synthase 1 is inactivated.

[0029] Technical Scheme 2 of the present invention provides a method for producing threonine by inactivating 2-methyl-citrate synthase 1 and enhancing the expression of at least one of aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase, and fructose-1,6-bisphosphatase 2.

[0030] Technical Scheme 3 of the present invention provides a method for producing threonine by obtaining and enhancing the expression of feedback inhibition-resistant aspartokinase and homoserine dehydrogenase, and inactivating 2-methyl-citrate synthase 1.

[0031] Technical Scheme 4 of the present invention provides a method for producing threonine by inactivating 2-methyl-citrate synthase 1 and enhancing the expression of aspartokinase, homoserine dehydrogenase and threonine synthase.

[0032] Technical Scheme 5 of the present invention provides a method for producing threonine by inactivating 2-methyl-citrate synthase 1 and enhancing the expression of aspartokinase, homoserine dehydrogenase and NAD kinase.

[0033] Technical Scheme 6 of the present invention provides a method for producing threonine by inactivating 2-methyl-citrate synthase 1 and enhancing the expression of aspartokinase, homoserine dehydrogenase, NAD kinase, and fructose-1,6-bisphosphatase 2.

[0034] Technical Scheme 7 of the present invention provides a method for producing threonine by inactivating 2-methyl-citrate synthase 1 and enhancing the expression of aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase, and fructose-1,6-diphosphatase.

[0035] The strain is Corynebacterium, preferably Corynebacterium glutamicum, and most preferably Corynebacterium glutamicum ATCC 13032.

[0036] The proteins according to the present invention and the genes encoding them are as follows. 2-Methyl-citrate synthase 1: coding gene name prpC1, NCBI number: cg0798, Cgl0696, NCgl0666. Aspartate aminotransferase: coding gene name aspB, NCBI number: cg0294, Cgl0240, NCgl0237. Aspartokinase: coding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247. Homoserine dehydrogenase: coding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136. Threonine synthase: coding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139. NAD kinase: coding gene name ppnK, NCBI number: cg1601, Cgl1413, NCgl1358. Fructose-1,6-bisphosphatase 2: coding gene name fbp / glpX, NCBI number: cg1157, Cgl1019, Ncgl0976.

[0037] The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise indicated, all examples are carried out according to standard laboratory conditions, such as those recommended in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in the manufacturer's instructions.

[0038] The experimental materials used in the following examples are as follows:

[0039] [Table A]

[0040] The experimental methods for the following examples are as follows.

[0041] The PCR amplification system is as follows.

[0042] [Table B]

[0043] The PCR amplification program is as follows:

[0044] [Table C]

[0045] Strain modification method: 1. Seamless assembly reaction program: Refer to the ClonExpress MultiS One Step Cloning Kit instructions. 2. Transformation method: Refer to the Trans1-T1 Phage Resistant Chemically Competent Cell instructions. 3. Preparation of competent cells: See C. glutamicum Handbook, Chapter 23.

[0046] Example 1 Construction of a plasmid for modifying the genome of a strain

[0047] 1. Aspartokinase Expression Enhancement Plasmid pK18mobsacB-P sod -lysC g1a-T311I Construction Using the Corynebacterium glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the P21 / P22 primer set to obtain the upstream homologous arm up, the P23 / P24 primer set to obtain the promoter fragment Psod, the P25 / P26 primer set to obtain lysCg1a-T311I, and the P27 / P28 primer set to obtain the downstream homologous arm dn. Using up and Psod as templates, fusion PCR was performed with the P21 / P24 primer set to obtain the fragment up-Psod. Using up-Psod, lysCg1a-T311I, and dn as templates, fusion PCR was performed with the P21 / P28 primer set to obtain the full-length fragment up-Psod-lysCg1a-T311I-dn. pK18mobsacB was digested with BamHI / HindIII. The two were assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I was obtained.

[0048] Here, g1a indicates that the first base of the start codon of the lysC gene (the wild-type lysC gene sequence is shown in SEQ ID NO:1) has been mutated from g to a, and T311I indicates that the 311th amino acid of aspartokinase encoded by the lysC gene has been mutated from T to I.

[0049] 2. Homoserine dehydrogenase expression-enhancing plasmid pK18mobsacB-P cspB -hom G378E Construction Using the Corynebacterium glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the P29 / P30 primer set to obtain the upstream homologous arm up, using the ATCC14067 genome as a template, PCR amplification was performed with the P31 / P32 primer set to obtain the promoter fragment PcspB, using the ATCC13032 genome as a template, PCR amplification was performed with the P33 / P34 primer set to obtain homG378E, and PCR amplification was performed with the P35 / P36 primer set to obtain the downstream homologous arm dn. Fusion PCR was performed with up and PcspB as templates, using the P29 / P32 primer set to obtain the fragment up-PcspB. Fusion PCR was performed with up-PcspB, homG378E, and dn as templates, using the P29 / P36 primer set to obtain the full-length fragment up-PcspB-homG378E-dn. pK18mobsacB was digested with BamHI / HindIII. The two were assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P cspB -hom G378E was obtained.

[0050] 3. Aspartate Aminothiocyanate Expression Enhancement Plasmid pK18mobsacB-P sod -Construction of aspB Using the Corynebacterium glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the P103 / P104 primer set to obtain the upstream homologous arm up, PCR amplification was performed with the P105 / P106 primer set to obtain the promoter fragment Psod, and PCR amplification was performed with the P107 / P108 primer set to obtain the downstream homologous arm dn. Fusion PCR was performed with the P103 / P108 primer set using up, Psod, and dn as templates to obtain the full-length fragment up-Psod-dn. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P sod -aspB was obtained.

[0051] 4. Threonine synthase expression-enhancing plasmid pK18mobsacB-P sod -thrC g1a Construction Using the ATCC13032 genome as a template, PCR amplification was performed with the P37 / P38 primer set to obtain the upstream homologous arm up, PCR amplification was performed with the P39 / P40 primer set to obtain the promoter fragment Psod-thrCg1a, and PCR amplification was performed with the P41 / P42 primer set to obtain the downstream homologous arm dn. Fusion PCR was performed with the P37 / P42 primer set using up, Psod-thrCV1M, and dn as templates to obtain the full-length fragment up-Psod-thrCg1a-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-P sod -thrC g1a was obtained.

[0052] Here, g1a indicates that the first base of the initiation codon of the thrC gene (the wild-type thrC gene sequence is shown in SEQ ID NO:2) has been mutated from g to a.

[0053] 5. Construction of 2-methyl-citrate synthase 1 inactivating plasmid pK18mobsacB-△prpC1 Using the Corynebacterium glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the prpC1-UF / prpC1-UR primer set to obtain the upstream homologous arm up, and PCR amplification was performed with the prpC1-DF / prpC1-DR primer set to obtain the downstream homologous arm dn. Fusion PCR was performed with the prpC1-UF / prpC1-DR primer set to obtain the full-length fragment up-dn using the up and dn templates. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit, and Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-△prpC1.

[0054] 6. NAD kinase expression enhancing plasmid pK18mobsacB-P tuf -Constructing ppnK Using the Corynebacterium glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the P109 / P110 primer set to obtain the upstream homologous arm up, PCR amplification was performed with the P111 / P112 primer set to obtain the promoter fragment Ptuf, and PCR amplification was performed with the P113 / P114 primer set to obtain the downstream homologous arm dn. Fusion PCR was performed with the P109 / P114 primer set using up, Ptuf, and dn as templates to obtain the full-length fragment up-Ptuf-dn. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P tuf -ppnK was obtained.

[0055] 7. Fructose-1,6-bisphosphatase Expression-enhancing Plasmid pK18mobsacB-P tuf -Build fbp Using the Corynebacterium glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the P61 / P62 primer set to obtain the upstream homologous arm up, PCR amplification was performed with the P63 / P64 primer set to obtain the promoter fragment Ptuf, and PCR amplification was performed with the P65 / P66 primer set to obtain the downstream homologous arm dn. Fusion PCR was performed with the P61 / P66 primer set using up, Ptuf, and dn as templates to obtain the full-length fragment up-Ptuf-dn. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P tuf -fbp was obtained.

[0056] The primers used in the construction process are shown in Table 1.

[0057] [Table 1]

[0058] Example 2 Construction of genome modified strain

[0059] 1. Construction of 2-methyl-citrate synthase 1 inactivated strain Corynebacterium glutamicum ATCC 13032 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-△prpC1 was transformed into the competent cells by electroporation, and the transformants in which the target gene was 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 standard liquid brain heart infusion medium, and the culture temperature was set 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 -2From 10 -4 The strain was serially diluted to 100% (100% dilution), 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 strain grown on sucrose medium did not carry the integrated vector sequence. The target sequence was amplified by PCR and subjected to nucleotide sequencing analysis, and the obtained target mutant was named SMCT089.

[0060] 2. Construction of Aspartokinase 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 -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 -4 The strain was serially diluted to 100% (100% dilution), 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 strain grown on sucrose medium did not carry the integrated vector sequence. The target sequence was amplified by PCR and subjected to nucleotide sequencing analysis, and the obtained target mutant was named SMCT076.

[0061] 3. Construction of homoserine dehydrogenase expression-enhanced strain SMCT076 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-P cspB -hom G378E 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 -4 The strain was serially diluted to 100% (100% dilution), 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 strain grown on sucrose medium did not carry the integrated vector sequence. The target sequence was amplified by PCR and subjected to nucleotide sequencing analysis, and the obtained target mutant strain was named SMCT077.

[0062] 4. Construction of Threonine Synthase Expression Enhanced Strain SMCT077 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-P sod -thrC g1atransformed 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 -4 The strain was serially diluted to 100% (100% dilution), 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 strain grown on sucrose medium did not carry the integrated vector sequence. The target sequence was amplified by PCR and subjected to nucleotide sequencing analysis, and the obtained target mutant strain was named SMCT078.

[0063] 5. Construction of NAD Kinase Expression Enhanced Strain SMCT077 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-P tuf The competent cells were transformed with -ppnK by electroporation, and transformants in which the target gene had been integrated into the chromosome by homology were selected in 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 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 100% (100% dilution), 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 strain grown on sucrose medium did not carry the integrated vector sequence. The target sequence was amplified by PCR and subjected to nucleotide sequencing analysis, and the obtained target mutant was named SMCT079.

[0064] 6. Construction of a strain with enhanced fructose-1,6-bisphosphatase expression SMCT077 and SMCT079 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). tuf For -fbp, the competent cells were transformed by electroporation, and transformants in which the target gene had been integrated into the chromosome by homology were selected in 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 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 -4 The strains were serially diluted to 100% (SMCT080, SMCT081, SMCT082, SMCT083, SMCT084, SMCT085, SMCT086, SMCT087, SMCT088, SMCT089, SMCT081, SMCT080, SMCT081). The diluted solutions were spread on standard solid brain heart infusion medium containing 10% sucrose and cultured at 33°C for 48 h. The genome of the strains grown on sucrose medium did not carry the integrated vector sequence. The target sequences were amplified by PCR and subjected to nucleotide sequencing analysis, and the obtained target mutants were named SMCT080 and SMCT081.

[0065] 7. Modification of SMCT081 by enhancing expression of aspartate aminotransferase and threonine synthase SMCT081 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 -4 The strains were serially diluted to 0.1 μg / ml, and the dilutions were applied to standard solid brain heart infusion medium containing 10% sucrose and cultured at 33°C for 48 h. The genomes of the strains grown on sucrose medium did not carry the integrated vector sequence. The target sequence was amplified by PCR, nucleotide sequencing analysis was performed, and the target mutant strains were obtained and competent cells were prepared. The recombinant plasmid pK18mobsacB-P sod -thrC g1a 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 strains were serially diluted to 100% (100% dilution), 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 strains grown on sucrose medium did not carry the integrated vector sequence. The target sequence was amplified by PCR and subjected to nucleotide sequencing analysis to obtain the target mutant strain SMCT082.

[0066] 8. Construction of 2-methyl-citrate synthase 1 inactivated strain SMCT077, SMCT078, SMCT079, SMCT080, SMCT081, and SMCT082 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-△prpC1 was transformed into the competent cells by electroporation, and the transformants in which the target gene was 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, and the culture temperature was set 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 -4 The mutants were serially diluted to 100% (SMCT083, SMCT084, SMCT085, SMCT086, SMCT087, and SMCT088) and the dilutions were spread onto standard solid brain heart infusion medium containing 10% sucrose and incubated at 33°C for 48 h. The genomes of strains grown on sucrose medium did not carry the integrated vector sequences. The target sequences were amplified by PCR and subjected to nucleotide sequencing analysis, and the obtained target mutants were named SMCT083, SMCT084, SMCT085, SMCT086, SMCT087, and SMCT088.

[0067] The strains obtained are shown in Table 2.

[0068] [Table 2]

[0069] Example 3 Shake flask validation of constructed strains

[0070] 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.

[0071] 2. L-Threonine production by shake flask fermentation of engineered fungi (1) Seed culture: A loopful of slant culture seed of SMCT076, SMCT077, SMCT078, SMCT079, SMCT080, SMCT081, SMCT082, SMCT083, SMCT084, SMCT085, SMCT086, and SMCT088 was taken and inoculated into a 500 mL Erlenmeyer flask containing 20 mL of seed medium, and cultured at 30°C and 220 r / min for 16 h with shaking. (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 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.

[0072] A comparison of the threonine production capacity of Corynebacterium glutamicum is shown in Table 3.

[0073] [Table 3]

[0074] As can be seen from Table 3, in the strain in which aspartokinase was modified based on the wild-type strain ATCC13032, the threonine production amount was initially accumulated, and with the enhanced expression of enzymes in the threonine synthesis pathway (homoserine dehydrogenase, aspartate aminotransferase, and threonine synthase), the threonine production amount was further improved. Subsequently, with the enhanced expression of NAD kinase and fructose 1,6-diphosphatase, the supply of reducing power of the strain was further enhanced, which is favorable for improving the threonine production amount.

[0075] In order to investigate the effect of 2-methyl-citrate synthase 1 on the production of threonine, 2-methyl-citrate synthase 1 was inactivated in the threonine-producing bacteria. As shown in Table 3, the production of threonine was improved to different degrees in all 2-methyl-citrate synthase 1 modified bacteria, and the highest improvement was 42% compared to the control strain. This shows that the inactivation of 2-methyl-citrate synthase 1 is advantageous for improving the production of threonine in the strain.

[0076] The above are merely preferred embodiments of the present invention, and those skilled in the art can make modifications and alterations without departing from the principles of the present invention (for example, to enhance expression, a strong promoter, a change in the RBS sequence, a change in the start codon, or an increase in activity by changing the amino acid sequence can be used, and to inactivate expression, inactivation or weakening of protein activity can be included), and all of these modifications and alterations are within the scope of the claims of the present invention.

Claims

1. A modified Corynebacterium microorganism, characterized in that the activity of 2-methyl-citrate synthase 1 is reduced or lost compared to an unmodified microorganism, and the modified microorganism has an improved threonine production amount compared to an unmodified microorganism.

2. The microorganism according to claim 1, characterized in that the reduction or loss of activity of 2-methyl-citrate synthase 1 in the microbial body is achieved by reducing expression of a gene encoding 2-methyl-citrate synthase 1 or by knocking out an endogenous gene encoding 2-methyl-citrate synthase 1.

3. 3. The microorganism according to claim 2, characterized in that the expression of the gene encoding 2-methyl-citrate synthase 1 is reduced or the endogenous gene encoding 2-methyl-citrate synthase 1 is knocked out using methods of mutagenesis, site-directed mutagenesis or homologous recombination.

4. The microorganism has enhanced activity of an enzyme involved in the threonine synthesis pathway in its body compared to an unmodified microorganism, The microorganism according to claim 1, characterized in that the enzyme related to the threonine synthesis pathway is selected from at least one of aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase, and fructose-1,6-diphosphatase 2.

5. The microorganism according to claim 4, characterized in that the microorganism is any one of the following (1) to (5). (1) A microorganism in which the activity of 2-methyl-citrate synthase 1 is reduced or absent and the activity of aspartokinase and / or homoserine dehydrogenase is enhanced. (2) A microorganism in which the activity of 2-methyl-citrate synthase 1 is reduced or lost, and the activities of aspartokinase, homoserine dehydrogenase, and / or threonine synthase are enhanced. (3) A microorganism in which the activity of 2-methyl-citrate synthase 1 is reduced or lost, and the activities of aspartokinase, homoserine dehydrogenase, and / or NAD kinase are enhanced. (4) A microorganism in which the activity of 2-methyl-citrate synthase 1 is reduced or lost, and the activities of aspartokinase, homoserine dehydrogenase, NAD kinase, and / or fructose-1,6-bisphosphatase 2 are enhanced. (5) A microorganism in which the activity of 2-methyl-citrate synthase 1 is decreased or absent, and the activities of aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase and fructose-1,6-bisphosphatase 2 are increased.

6. The microorganism according to claim 4, characterized in that the enhancement of the activity of an enzyme involved in the threonine synthesis pathway in the microbial body 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

7. The microorganism according to any one of claims 1 to 6, characterized in that the microorganism is Corynebacterium glutamicum.

8. A. Attenuating the gene encoding 2-methyl-citrate synthase 1 in an amino acid-producing Corynebacterium to obtain a gene-attenuated strain, and B. enhancing the enzymes involved in the threonine synthesis pathway in the gene-weakened strain obtained in step A to obtain a strain with enhanced enzyme activity; The attenuation comprises knocking out or reducing the expression of a gene encoding 2-methyl-citrate synthase 1; The enhancement is selected from the following 1) to 5) 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 A method for constructing a threonine-producing engineered strain, characterized in that the enzyme involved in the threonine synthesis pathway is selected from at least one of aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine synthase, NAD kinase, and fructose-1,6-diphosphatase 2.

9. 9. The method of claim 8, wherein the Corynebacterium is Corynebacterium glutamicum.

10. a) culturing a microorganism according to any one of claims 1 to 7 or an engineered fungus constructed by the method according to claim 8 or 9, and obtaining a culture of said microorganism or engineered fungus; b) recovering threonine from the culture obtained in step a).

Images