WO2023151421A1 - Modified corynebacterium microorganism, and use thereof and construction method therefor - Google Patents

Abstract

Provided are a modified Corynebacterium microorganism, a construction method therefor, and the use thereof in the production of threonine. The modified Corynebacterium microorganism has a weakened or inactivated expression of Cgl0978 gene compared with unmodified microorganisms, and has an enhanced production capability for threonine and a reduced yield of isoleucine compared with the unmodified microorganisms.

Description

A modified Corynebacterium microorganism and its application and construction method technical field

The invention relates to the technical field of microbial engineering, in particular to a modified microorganism of the genus Corynebacterium and its application and construction method.

Background technique

L-threonine (L-Threonin), the chemical name is β-hydroxy-α-aminobutyric acid, the molecular formula is C ₄ H ₉ NO ₃ , and the relative molecular mass is 119.12. L-threonine is an essential amino acid. Threonine is mainly used in medicine, chemical reagents, food fortifiers, feed additives, etc.

In Corynebacterium glutamicum, the generation of threonine from oxaloacetate requires five steps of catalytic reactions, which are aspartate kinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), and homoserine dehydrogenase (encoded by asd). Hydrogenase (encoded by hom), homoserine kinase (encoded by thrB) and threonine synthase (encoded by thrC). Hermann Sahm et al. have been working on the development of a high-threonine-yielding glutamicum strain, and have made a breakthrough, obtaining a hom gene that is resistant to feedback inhibition (Reinscheid D J, Eikmanns B J, 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.), lysC gene (Eikmanns B J,Eggeling L,Sahm H.Molecular aspects of lysine,threonine, and isoleucine biosynthesis in Corynebacterium glutamicum.[J].Antonie Van Leeuwenhoek,1993,64(2):145-163.). Following Hermann Sahm, Lothar Eggling carried out further exploration in this field, weakening the coding gene glyA in the threonine utilization pathway, and overexpressing the threonine export protein ThrE, which increased the threonine production from 49mM to 67mM (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.).

However, the current reports on the production of threonine by Corynebacterium glutamicum mainly focus on the deregulation and overexpression of its synthetic pathway, and there are few reports on the TCA cycle and central metabolism. Moreover, the existing reports have only done preliminary research on the synthesis pathway of threonine, and have not formed a system. Further studies on threonine production by Corynebacterium glutamicum are still necessary.

Contents of the invention

The purpose of the present invention is to improve the ability of the strain to produce threonine by inactivating or weakening the expression of the Cgl0978 gene, thereby providing a threonine (L-threonine) producing strain and its construction method and application.

In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a modified microorganism of the genus Corynebacterium, the expression of the Cgl0978 gene of the microorganism is reduced or lost compared with the unmodified microorganism, and the The modified microorganism has enhanced threonine production capacity.

The same genes as the Cgl0978 gene also have genes numbered NCgl0939 and cg1116.

At present, there are few studies on the degradation pathway of threonine. Threonine is catalyzed by threonine dehydratase to generate isoleucine. At present, the gene encoding this enzyme is ilvA, and the mainstream is to reduce the by-product of isoleucine. The method is to reduce the expression level of ilvA, or inactivate ilvA. The research of the present invention finds that Cgl0978 has the function of threonine dehydratase, and the production of isoleucine is reduced by inactivating Cgl0978, and the ability of the bacterial strain to synthesize threonine is improved.

Mutagenesis, site-directed mutation or homologous recombination can be used to reduce or inactivate the expression of the Cgl0978 gene (such as knocking out the endogenous Cgl0978 gene).

Further, compared with unmodified microorganisms, the activity of enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the microorganism is enhanced; wherein, the threonine synthesis pathway and/or precursor Enzymes related to the supply pathway are selected from at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase; preferably, their reference sequences on NCBI The numbers are respectively WP_003855724.1, WP_003854900.1, WP_011013816.1, WP_011014465.1, or amino acid sequences with a similarity of 90% to the above reference sequence.

Preferably, the microorganism is any one of the following ①～④:

① Microorganisms with reduced or lost expression of the Cgl0978 gene and enhanced aspartokinase and/or homoserine dehydrogenase activities;

② Microorganisms with reduced or lost expression of Cgl0978 gene and enhanced activities of aspartokinase, homoserine dehydrogenase and/or pyruvate carboxylase;

③ Microorganisms with reduced or lost expression of Cgl0978 gene and enhanced activity of aspartokinase, homoserine dehydrogenase and/or phosphoenolpyruvate carboxylase;

④ Microorganisms with reduced or lost expression of Cgl0978 gene and enhanced activities of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase and/or phosphoenolpyruvate carboxylase.

The enhancement of the activity of enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the microorganism is achieved by being selected from the following 1) to 5), or an optional combination:

1) enhanced by introducing a plasmid having a gene encoding the enzyme;

2) enhanced by increasing the copy number of the gene encoding said enzyme on the chromosome;

3) Enhanced by changing the promoter sequence of the gene encoding the enzyme on the chromosome;

4) Enhanced by operably linking a strong promoter to the gene encoding the enzyme;

5) Enhanced by changing the amino acid sequence of the enzyme.

Preferably, the Corynebacterium glutamicum described in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum evolutionary tree https:/ /www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.

In a second aspect, the present invention provides a method for constructing a threonine-producing strain, the method comprising:

A. Weaken or inactivate the Cgl0978 gene in corynebacteria with amino acid production ability to obtain gene weakened strains; and/or,

B. Enhancing the enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the gene-weakened strain of step A to obtain a strain with enhanced enzyme activity;

The enhanced approach is selected from the following 1) to 5), or an optional combination:

1) enhanced by introducing a plasmid having a gene encoding the enzyme;

2) enhanced by increasing the copy number of the gene encoding said enzyme on the chromosome;

3) Enhanced by changing the promoter sequence of the gene encoding the enzyme on the chromosome;

4) Enhanced by operably linking a strong promoter to the gene encoding the enzyme;

5) Enhanced by changing the amino acid sequence of the enzyme;

Wherein, the enzymes related to the threonine synthesis pathway and/or precursor supply pathway are selected from aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, phosphoenol pyruvate carboxylase at least one of .

In a third aspect, the present invention provides a method for producing threonine, the method comprising the steps of:

a) cultivating the above-mentioned microorganisms to obtain a culture of said microorganisms;

b) collecting the threonine produced from said culture obtained in step a).

In the fourth aspect, the present invention provides the application of Cgl0978 gene attenuation or inactivation in threonine fermentation production or improvement of threonine fermentation yield.

Further, the fermentation yield of threonine is improved by inactivating the Cgl0978 gene in Corynebacterium having amino acid production ability.

Preferably, the Corynebacterium glutamicum described in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum evolutionary tree https:/ /www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.

In a fifth aspect, the present invention provides the use of the modified Corynebacterium genus microorganism or the threonine-producing strain constructed according to the above-mentioned method in the fermentative production of threonine or in improving the fermentative yield of threonine.

The transformation methods of the above-mentioned related strains, including gene enhancement and weakening, are transformation methods known to those skilled in the art. ,2016; Cui Yi. Metabolic engineering of Corynebacterium glutamicum to produce L--leucine [D]. Tianjin University of Science and Technology.; Xu Guodong. Construction of L-isoleucine production strain and optimization of fermentation conditions. Tianjin University of Science and Technology ,2015.

Preferably, in the present invention, the Cgl0978 coding region is inactivated by removing it from the genome.

By mutating the gene lysC encoding aspartokinase, its start codon is mutated from GTG to ATG, the 311th amino acid encoded by it is changed from threonine to isoleucine, and the lysC gene is changed from Psod Initiates transcription, culminating in expression enhancement and deregulation of aspartokinase. The nucleotide sequence of Psod is shown in SEQ ID NO.37.

By mutating the gene hom encoding homoserine dehydrogenase so that its encoded protein carries the G378E mutation, and enabling the transcription of the hom gene to be initiated by PcspB, the deregulation and expression enhancement of homoserine dehydrogenase are finally realized. The nucleotide sequence of PcspB is shown in SEQ ID NO.38.

By mutating the gene pyc encoding pyruvate carboxylase so that its encoded protein carries the P458S mutation, and making the pyc gene be transcribed by Psod, the expression enhancement of pyruvate carboxylase is finally realized. The nucleotide sequence of Psod is shown in SEQ ID NO.37.

By mutating the gene ppc encoding phosphoenolpyruvate carboxylase, so that the encoded protein carries the D299N mutation, and the transcription of the ppc gene is initiated by P _tuf , the expression enhancement of phosphoenolpyruvate carboxylase is finally realized . The nucleotide sequence of P _tuf is shown in SEQ ID NO.39.

The beneficial effects of the present invention are at least:

In the present invention, the Cgl0978 inactivated strain is applied to threonine production, and the threonine yield can be increased by 20.8-51.2% compared with that before the modification, and the isoleucine content can be reduced from 0.8g/L to 0.2g/L. Specifically, the inactivated Cgl0978 is further enhanced and reconciled with the expression of at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase in the threonine synthesis pathway. When the regulation is combined, the yield of threonine is increased, and the yield of isoleucine, a downstream product of threonine, is decreased, which provides a new idea for improving the production capacity of threonine.

Detailed ways

Preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given for the purpose of illustration only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The proteins involved in the present invention and their coding genes are as follows:

Aspartokinase, encoded gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247;

Homoserine dehydrogenase, encoding gene name hom, NCBI number: Cg1337, Cgl1183, NCgl1136;

Threonine synthase, encoding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139;

Pyruvate carboxylase, encoding gene pyc, NCBI number: cg0791, Cgl0689, NCgl0659;

Phosphoenolpyruvate carboxylase, encoding gene ppc, NCBI number: cg1787, Cgl1585, NCgl1523.

In the present invention, the model strain ATCC13032 was used as the starting strain to construct the inactivated strain of Cgl0978, and it was found that the inactivation of Cgl0978 had no effect on the strain. Since the model strain ATCC13032 is not a threonine-producing bacterium, it is foreseeable that the inactivation of Cgl0978 has no obvious effect on the strain of.

In order to explore whether the inactivation of Cgl0978 will reduce the ability of threonine to be degraded into isoleucine, the present invention firstly constructed a strain with threonine production capacity, first deregulated and strengthened the expression of aspartokinase, and then By deregulating and enhancing the expression of homoserine dehydrogenase, the modified strain SMCT363 with threonine production capacity was obtained. The threonine yield of SMCT363 was 2.4g/L, and the isoleucine content was 0.3g/L. The threonine yield of the modified strain SMCT364 obtained by inactivating Cgl0978 of SMCT363 was 2.9 g/L, the isoleucine content was 0.1 g/L, the isoleucine content was reduced, and the threonine yield was increased by 20.8%.

In order to further verify that Cgl0978 can reduce the degradation of threonine to isoleucine, threonine-producing strains SMCT365 and SMCT366 were further constructed, which respectively express enhanced and deregulated pyruvate carboxylase and phosphoenolpyruvate on the basis of SMCT363 carboxylase. And further inactivated Cgl0978 to obtain modified strains SMCT367 and SMCT368, the threonine yields of the modified strains increased by 30% and 41.2% respectively compared with those without inactivation of Cgl0978.

Finally, on the basis of SMCT366, the modified strain SMCT369 was obtained by strengthening and deregulating phosphoenolpyruvate carboxylase, and then Cgl0978 was inactivated to obtain SMCT370. g/L.

Inactivation or weakening during the transformation process includes promoter replacement, ribosome binding site changes, point mutations, and sequence deletions. Expression enhancement during transformation includes promoter replacement and ribosome binding site changes. , copy number increase, plasmid overexpression and other means, and the above means are well known to researchers in the field. The above means cannot be exhausted by examples, so the embodiments of the present invention only use promoter enhancement and point mutations as representatives for illustration.

Example 1 Strain Genome Modification Plasmid Construction

1) Cgl0978 inactivation scheme recombinant plasmid pK18mobsacB-△Cgl0978

Using the ATCC13032 genome as a template, the upstream homology arm up was obtained by PCR amplification with the P145/P146 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P147/P148 primer pair. Fusion PCR was performed as a template to obtain the full-length fragment △Cgl0978. pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, transformed into Trans1T1 competent cells, and obtained the recombinant plasmid pK18mobsacB-△Cgl0978

2) Recombinant plasmid pK18mobsacB-P _sod -lysC ^g1a-T311I aspartokinase expression enhancement and deregulation scheme

Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, the upstream homology arm up was obtained by PCR amplification with the P21/P22 primer pair, the promoter fragment Psod was obtained by PCR amplification with the P23/P24 primer pair, and the P25/P26 primer The pair was amplified by PCR to obtain lysC ^g1a-T311I , and the downstream homology arm dn was obtained by PCR amplification with the P27/P28 primer pair. Fusion PCR was carried out with P21/P24 primer pair and up and Psod as templates to obtain the fragment up-Psod. The full-length fragment up-Psod-lysC ^g1a-T311I -dn was obtained by fusion PCR with P21/P28 primer pair and up-Psod, lysC ^g1a -T311I , dn as templates. pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P _sod -lysC ^g1a-T311I .

3) Recombinant plasmid pK18mobsacB-P _cspB -hom ^G378E for homoserine dehydrogenase expression enhancement and deregulation scheme

Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed with the P29/P30 primer pair to obtain the upstream homology arm up, and the ATCC14067 genome was used as a template to perform PCR amplification with the P31/P32 primer pair to obtain the promoter fragment PcspB, The ATCC13032 genome was used as a template to obtain hom ^G378E by PCR amplification with P33/P34 primer pair, and the downstream homology arm dn was obtained by PCR amplification with P35/P36 primer pair. Fusion PCR was carried out with P29/P32 primer pair and up and PcspB as templates to obtain the fragment up-PcspB. The full-length fragment up-PcspB-hom ^G378E -dn was obtained by fusion PCR with P29/P36 primer pair and up-PcspB, hom ^G378E , dn as template. pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P _cspB -hom ^G378E .

4) Pyruvate carboxylase expression enhancement and deregulation program recombinant plasmid pK18mobsacB-P _sod -pyc ^P458S

Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, the upstream homology arm up was obtained by PCR amplification with the P13/P14 primer pair, the promoter fragment Psod was obtained by PCR amplification with the P15/P16 primer pair, and the P17/P18 primer The pair was amplified by PCR to obtain pyc ^P458S , and the downstream homology arm dn was obtained by PCR amplification with the P19/P20 primer pair. Using the P13/P16 primer pair to perform fusion PCR with up and Psod as templates, the fragment up-Psod was obtained. Fusion PCR was performed with P13/P20 primer pair and up-Psod, pyc ^P458S and dn as templates to obtain the full-length fragment up-Psod-pyc ^P458S -dn. pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P _sod -pyc ^P458S .

5) Phosphoenolpyruvate carboxylase expression enhancement and deregulation program recombinant plasmid pK18mobsacB-P _tuf -ppc ^D299N

Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, the upstream homology arm up was obtained by PCR amplification with the P53/P54 primer pair, the promoter fragment Ptuf was obtained by PCR amplification with the P55/P56 primer pair, and the P57/P58 primer The ppc ^D299N was obtained by PCR amplification, and the downstream homology arm dn was obtained by PCR amplification with the P59/P60 primer pair. Fusion PCR was performed with P53/P56 primer pair and up and Ptuf as templates to obtain the fragment up-Ptuf. The full-length fragment up-Ptuf-ppc ^D299N -dn was obtained by fusion PCR with P53/P60 primer pair and up-Ptuf, ppc ^D299N and dn as templates. pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P _tuf -ppc ^D299N .

The primers used in the plasmid construction process are shown in Table 1 below:

Table 1

name Sequence (SEQ ID No.1-36) P13 AATTCGAGCTCGGTACCCGGGGATCCTGACAGTTGCTGATCTGGCT P14 CCCGGAATAATTGGCAGCTATAGAGTAATTTTCCTTTCA P15 TGAAAGGAATAATTACTCTATAGCTGCCAATTATTCCGGG P16 GAAGATGTGTGAGTCGACACGGGTAAAAAATCCTTTCGTA P17 TACGAAAGGATTTTTTACCCGTGTCGACTCACACATCTTC P18 GGTGGAGCCTGAAGGAGGTGCGAGTGATCGGCAATGAATCCGG P19 CCGGATTCATTGCCGATCACTCGCACCTCCTTCAGGCTCCACC P20 GTAAAACGACGGCCAGTGCCAAGCTTCGCGGCAGACGGAGTCTGGG P21 AATTCGAGCTCGGTACCCGGGGATCCAGCGACAGGACAAGCACTGG P22 CCCGGAATAATTGGCAGCTATGTGCACCTTTCGATCTACG P23 CGTAGATCGAAAGGTGCACATAGCTGCCAATTATTCCGGG P24 TTTCTGTACGACCAGGGCCATGGGTAAAAAATCCTTTCGTA P25 TACGAAAGGATTTTTTACCCATGGCCCTGGTCGTACAGAAA P26 TCGGAACGAGGGCAGGTGAAGGTGATGTCGGTGGTGCCGTCT P27 AGACGGCACCACCGACATCACCTTCACCTGCCCTCGTTCCGA P28 GTAAAACGACGGCCAGTGCCAAGCTTAGCCTGGTAAGAGGAAACGT P29 AATTCGAGCTCGGTACCCGGGGATCCCTGCGGGCAGATCCTTTTGA P30 ATTTCTTTATAAACGCAGGTCATATCTACCAAAAACTACGC P31 GCGTAGTTTTGGTAGATATGACCTGCGTTTATAAAGAAAT P32 GTATATCTCTTCTGCAGGAATAGGTATCGAAAGACGAAA P33 TTTCGTCTTTCGATACCTATTCCTGCAGAAGGAGATATAC

P34 TAGCCAATTCAGCCAAAACCCCCACGCGATCTTCCACATCC P35 GGATGTGGAAGATCGCGTGGGGGTTTTGGCTGAATTGGCTA P36 GTAAAACGACGGCCAGTGCCAAGCTTGCTGGCTCTTGCCGTCGATA P53 AATTCGAGCTCGGTACCCGGGGATCCTACGTCGTCGAGCAGACCCG P54 CATTCGCAGGGTAACGGCCAAGGGTGTTGGCGTGCATGAG P55 CTCATGCACGCCAACACCCTTGGCCGTTACCCTGCGAATG P56 TCGCGTAAAAAATCAGTCATTGTATGTCCTCCTGGACTTC P57 GAAGTCCAGGAGGACATACAATGACTGATTTTTTACGCGA P58 GTGACCTTATTCATGCGGTTCGACAGGCTGAGCTCATGCT P59 AGCATGAGCTCAGCCTGTCGAACCGCATGAATAAGGTCAC P60 GTAAAACGACGGCCAGTGCCAAGCTTGGTGACTTGGGCGCGTTCGA P145 AATTCGAGCTCGGTACCCGGGGATCCGCAGGCACCTTCACCACAAT P146 CATCGAGTTCTAGAAAACACAGGCTGTGATTTCAAACGATCACACGACG P147 CGTCGTGTGATCGTTTGAAATCACAGCTGTGTTTTCTAGAACTCGATG P148 CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTGCATCCAAACGCTTCCATCTTC

The construction of embodiment 2 genome modification bacterial strains

1) Construction of Aspartokinase Expression Enhanced and Deregulated Strains

ATCC13032 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-Psod-lysC ^g1a-T311I was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg/L kanamycin, in which the target gene was inserted into the chromosome due to homology middle. The screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange. The culture was serially diluted (from 10 ^-2 to 10 ^-4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strains were obtained and named SMCT362 respectively. In this strain, the lysC gene was mutated, its start codon was mutated from GTG to ATG, the 311th amino acid encoded by it was changed from threonine to isoleucine, and the promoter of lysC gene was replaced with a strong promoter Psod.

2) Construction of homoserine dehydrogenase expression enhanced and deregulated strains

For the strain construction method, refer to the above 1), using SMCT362 as the starting strain, carry out the modification of homoserine dehydrogenase expression enhancement and deregulation (pK18mobsacB-P _cspB -hom ^G378E is introduced into SMCT362), and the obtained modified strain is named SMCT363. In this strain, the hom gene was further mutated, and the corresponding amino acid mutation site was G378E, and the promoter of the hom gene was replaced with a strong promoter PcspB.

3) Construction of Pyruvate Carboxylase Expression Enhanced and Deregulated Strains

For the strain construction method, refer to the above 1), SMCT363 was used as the starting bacteria to carry out the modification of pyruvate carboxylase expression enhancement and deregulation (pK18mobsacB-P _sod -pyc ^P458S was introduced into SMCT363), and the obtained modified strain was named SMCT365. In this strain, the pyc gene is further mutated, the corresponding amino acid mutation site is P458S, and the promoter of the pyc gene is replaced by a strong promoter P _sod .

4) Construction of phosphoenolpyruvate carboxylase expression strong and deregulated strain

The strain construction method refers to the above 1), using SMCT363 and SMCT365 as the starting bacteria, the transformation of phosphoenolpyruvate carboxylase expression enhancement and deregulation (introducing pK18mobsacB-P _tuf -ppc ^D299N into SMCT363 and SMCT365), the obtained transformation The strains were named SMCT366, SMCT369. In this strain, the ppc gene was further mutated, the corresponding amino acid mutation site was D299N, and the promoter of the ppc gene was replaced with a strong promoter P _tuf .

5) Construction of Cgl0978 inactivated strain

Refer to the above 1) for the strain construction method, use ATCC13032, SMCT363, SMCT365, SMCT366, SMCT369 as the starting bacteria, carry out the transformation of the Cgl0978 inactivated strain (introduce pK18mobsacB-△Cgl0978 into the above starting bacteria), and the obtained modified strains are named SMCT361, SMCT364 , SMCT367, SMCT368, SMCT370. The coding region of the Cgl0978 gene was lost in this strain, resulting in its inactivation.

The list of strains obtained is shown in Table 2 below.

Table 2

strain genotype SMCT361 ATCC13032,ΔCgl0978 SMCT362 ATCC13032, P _sod -lysC ^g1a-T311I SMCT363 ATCC13032, P _sod -lysC ^g1a-T311I , P _cspB -hom ^G378E SMCT364 ATCC13032, P _sod -lysC ^g1a-T311I , P _cspB -hom ^G378E ,ΔCgl0978 SMCT365 ATCC13032, P _sod -lysC ^g1a-T311I , P _cspB -hom ^G378E , P _sod -pyc ^P458S SMCT366 ATCC13032, P _sod -lysC ^g1a-T311I , P _cspB -hom ^G378E , P _tuf -ppc ^D299N SMCT367 ATCC13032,P _sod -lysC ^g1a-T311I ,P _cspB -hom ^G378E ,P _sod -pyc ^P458S ,ΔCgl0978 SMCT368 ATCC13032, P _sod -lysC ^g1a-T311I , P _cspB -hom ^G378E , P _tuf -ppc ^D299N , ΔCgl0978 SMCT369 ATCC13032,P _sod -lysC ^g1a-T311I ,P _cspB -hom ^G378E ,P _sod -pyc ^P458S ,P _tuf -ppc ^D299N SMCT370 ATCC13032, P _sod -lysC ^g1a-T311I , P _cspB -hom ^G378E , P _sod -pyc ^P458S , P _tuf -ppc ^D299N , ΔCgl0978

Embodiment 3 constructs bacterial strain shaking flask verification

1. Medium

Seed activation medium: BHI 3.7%, agar 2%, pH7.

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.

2. Shake flask fermentation of engineering bacteria to produce L-threonine

(1) Seed culture: Pick ATCC13032, SMCT361, SMCT363, SMCT364, SMCT365, SMCT366, SMCT367, SMCT368, SMCT369, SMCT370 slant seeds 1 ring and connect them to a 500mL Erlenmeyer flask containing 20mL seed medium, 30°C, 220r/ Min shake culture 16h.

(2) Fermentation culture: inoculate 2 mL of seed solution into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture at 33° C. and 220 r/min for 24 hours with shaking.

(3) Take 1 mL of fermentation broth and centrifuge (12000rpm, 2min), collect the supernatant, and use HPLC to detect the concentration of L-threonine and isoleucine in the fermentation broth of engineering bacteria and control bacteria.

(4) Threonine shake flask fermentation results are shown in Table 3 below.

table 3

It can be seen from the above table that the threonine yield of the modified strain after the inactivation of Cgl0978 is increased compared with that before the inactivation, and the yield is increased between 20.8% and 51.2%, and the highest threonine yield is 6.5g/L. The threonine yields of different Cgl0978 inactivation strains are different, ranging from 0.9g/L to 3.6g/L, indicating that the inactivation of Cgl0978 and the combination of different sites have different effects, and when it is combined with When at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase in the threonine synthesis pathway is enhanced and deregulated, the threonine The yields were all increased by 34% to 124%.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (9)

A modified microorganism of the genus Corynebacterium, wherein, compared with an unmodified microorganism, the expression of the Cgl0978 gene of the microorganism is reduced or lost, and the microorganism has an enhanced threonine production ability compared with the unmodified microorganism . The microorganism according to claim 1, wherein the expression of the Cgl0978 gene is reduced or inactivated by mutagenesis, site-directed mutation or homologous recombination. The microorganism according to claim 1, wherein, compared with the unmodified microorganism, the activity of enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the microorganism is enhanced; Wherein, the enzymes related to the threonine synthesis pathway and/or precursor supply pathway are selected from aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxylase at least one. The microorganism according to claim 3, wherein the microorganism is any one of the following ① to ④: ① Microorganisms with reduced or lost expression of the Cgl0978 gene and enhanced aspartokinase and/or homoserine dehydrogenase activities; ② Microorganisms with reduced or lost expression of Cgl0978 gene and enhanced activities of aspartokinase, homoserine dehydrogenase and/or pyruvate carboxylase; ③ Microorganisms with reduced or lost expression of Cgl0978 gene and enhanced activity of aspartokinase, homoserine dehydrogenase and/or phosphoenolpyruvate carboxylase; ④ Microorganisms with reduced or lost expression of Cgl0978 gene and enhanced activities of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase and/or phosphoenolpyruvate carboxylase. The microorganism according to claim 3, wherein, the enhancement of the activity of enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the microorganism is selected from the following 1) to 5), or an optional combination Achieved: 1) enhanced by introducing a plasmid having a gene encoding the enzyme; 2) enhanced by increasing the copy number of the gene encoding said enzyme on the chromosome; 3) Enhanced by changing the promoter sequence of the gene encoding the enzyme on the chromosome; 4) Enhanced by operably linking a strong promoter to the gene encoding the enzyme; 5) Enhanced by changing the amino acid sequence of the enzyme. The microorganism according to any one of claims 1 to 5, wherein the microorganism is Corynebacterium glutamicum. A method for constructing a threonine-producing bacterial strain, wherein the method comprises: A. Weaken or inactivate the Cgl0978 gene in coryneform bacteria with amino acid production ability to obtain gene weakened strains; and optionally B. Enhancing the enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the gene weakened strain in step A to obtain enzyme activity enhanced strains; The enhanced pathway is selected from the following 1) to 5), or an optional combination: 1) enhanced by introducing a plasmid having a gene encoding the enzyme; 2) enhanced by increasing the copy number of the gene encoding said enzyme on the chromosome; 3) Enhanced by changing the promoter sequence of the gene encoding the enzyme on the chromosome; 4) Enhanced by operably linking a strong promoter to the gene encoding the enzyme; 5) enhanced by changing the amino acid sequence of the enzyme; Wherein, the enzymes related to the threonine synthesis pathway and/or precursor supply pathway are selected from aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxylase at least one. The method according to claim 7, wherein the corynebacterium is Corynebacterium glutamicum. A method for producing threonine, wherein the method comprises the steps of: A) cultivating the microorganism described in any one of claims 1 to 6 or the bacterial strain constructed by the method described in claim 7 or 8, to obtain the culture or bacterial strain of said microorganism; b) collecting the threonine produced from said culture obtained in step a).