CN116536227A - Threonine-producing modified corynebacterium microorganism, construction method and application thereof - Google Patents

Threonine-producing modified corynebacterium microorganism, construction method and application thereof Download PDF

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CN116536227A
CN116536227A CN202210096136.1A CN202210096136A CN116536227A CN 116536227 A CN116536227 A CN 116536227A CN 202210096136 A CN202210096136 A CN 202210096136A CN 116536227 A CN116536227 A CN 116536227A
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enzyme
microorganism
threonine
enhanced
gene encoding
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康培
程江红
宫卫波
何君
李岩
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Langfang Meihua Bio Technology Development Co Ltd
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Abstract

The invention relates to the technical field of microbial engineering, and in particular discloses a threonine-producing modified corynebacterium microorganism, and a construction method and application thereof. The modified coryneform microorganism of the present invention has reduced or lost malic enzyme activity as compared to an unmodified microorganism, and the microorganism has enhanced threonine productivity as compared to an unmodified microorganism. The recombinant microorganism of the present invention can enhance threonine production. Provides a new way for large-scale production of threonine.

Description

Threonine-producing modified corynebacterium microorganism, construction method and application thereof
Technical Field
The invention relates to the technical field of microbial engineering, in particular to a threonine-producing modified corynebacterium microorganism, and a construction method and application thereof.
Background
L-Threonine (L-Threonine), chemical name of beta-hydroxy-alpha-aminobutyric acid, molecular formula of C 4 H 9 NO 3 The relative molecular mass was 119.12. L-threonine is an essential amino acid, and threonine is mainly used in medicine, chemical reagents, food enhancers, feed additives and the like.
In Corynebacterium glutamicum, five catalytic reactions, aspartokinase (lysC-encoded), aspartate semialdehyde dehydrogenase (asd-encoded), homoserine dehydrogenase (hom-encoded), homoserine kinase (thrB-encoded) and threonine synthase (thrC-encoded), are required for threonine production from oxaloacetic acid. Hermann Sahm et al have been working on the development of high threonine producing cereal strains and have made some breakthroughs to obtain the hom gene against 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.), the 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 samm, lothar Eggling has further explored in the field to attenuate the coding gene glyA in the threonine-utilizing pathway, while overexpressing threonine-carrying protein ThrE, so that threonine production increases 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 report of threonine production by corynebacterium glutamicum is mainly focused on the synthesis route, few reports about precursor supply and the like are provided, no report of adjusting the anaplerotic route between glycolysis and TCA cycle to improve threonine yield is provided (the route has no direct relation with threonine synthesis), and the existing report only makes preliminary researches on the threonine synthesis route and does not form a system.
Therefore, further studies on threonine production by Corynebacterium glutamicum are necessary.
Disclosure of Invention
The invention aims to improve the threonine producing capacity of a strain by inactivating malic enzyme, thereby providing a threonine (L-threonine) producing strain, a construction method and application thereof.
To achieve the object of the present invention, in a first aspect, the present invention provides a modified microorganism of the genus Corynebacterium, which has reduced or lost malic enzyme activity as compared to an unmodified microorganism and which has enhanced threonine productivity as compared to an unmodified microorganism. Preferably, the malic enzyme has a reference sequence number wp_011015562.1 on NCBI, or an amino acid sequence with a similarity of 90%.
Further, the reduction or loss of the activity of the malic enzyme in the microorganism is achieved by reducing the expression of the gene encoding the malic enzyme or knocking out the endogenous gene encoding the malic enzyme.
Mutagenesis, site-directed mutagenesis or homologous recombination may be used to reduce expression of the gene encoding the malic enzyme or to knock out the endogenous gene encoding the malic enzyme.
Further, the microorganism has an increased activity of an enzyme involved in threonine synthesis and/or precursor supply pathway in vivo as compared to the unmodified microorganism; wherein the enzyme associated with the threonine synthesis and/or precursor supply pathway is selected from at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxylase; preferably, their reference sequence numbers at NCBI are wp_003855724.1, wp_003854900.1, wp_011013816.1, wp_011014465.1, respectively, or amino acid sequences with a similarity of 90% with the above reference sequences.
Preferably, the microorganism is any one of the following (1) to (4):
(1) a microorganism having reduced or lost malate activity and enhanced activity of an aspartate kinase and/or a homoserine dehydrogenase;
(2) a microorganism having reduced or lost malate activity and enhanced aspartokinase, homoserine dehydrogenase and/or pyruvate carboxylase activity;
(3) a microorganism having reduced or lost malate activity and enhanced aspartokinase, homoserine dehydrogenase and/or phosphoenolpyruvate carboxylase activity;
(4) a microorganism having reduced or lost malate enzyme activity and enhanced aspartokinase, homoserine dehydrogenase, pyruvate carboxylase and/or phosphoenolpyruvate carboxylase activity.
The enhancement of threonine synthesis and/or pre-supply of the activity of the pathway-related enzymes in the microorganism is achieved by a compound selected from the following 1) to 6), or optionally:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by altering the nucleotide sequence encoding the enzyme gene.
Preferably, the corynebacterium of the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum, 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. weakening a gene encoding malic enzyme in coryneform bacteria having amino acid production ability to obtain a gene-weakened strain; the attenuation includes knocking out or reducing expression of a gene encoding a malic enzyme; and/or
B. Enhancing the enzyme related to threonine synthesis and/or precursor supply paths in the gene-attenuated strain of the step A to obtain an enzyme activity-enhanced strain;
the enhanced pathway is selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhancement by altering the nucleotide sequence encoding the enzyme gene;
wherein the enzyme associated with the threonine synthesis and/or precursor supply pathway is selected from at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxylase.
In a third aspect, the present invention provides a method for producing threonine, the method comprising the steps of:
a) Culturing the microorganism to obtain a culture of the microorganism;
b) Collecting the threonine produced from the culture obtained in step a).
In a fourth aspect, the invention provides the use of a knockout or reduced expression of a gene encoding a malic enzyme in threonine fermentation production or for increasing threonine fermentation production.
Further, the fermentation yield of threonine is improved by inactivating the malic enzyme in coryneform bacteria (Corynebacterium) having an amino acid-producing ability.
Preferably, the corynebacterium of the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum, 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 coryneform microorganism or the threonine-producing strain constructed according to the above-mentioned method for threonine fermentation production or for improving threonine fermentation production.
The transformation methods of the related strains comprise transformation modes of strengthening and weakening genes and the like which are known to the person skilled in the art, and are referred to the system path engineering of the full-scope high-yield L-arginine corynebacterium crenatum [ D ]. University of Jiangnan, 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-producing Strain and optimization of fermentation conditions university of Tianjin science and technology 2015.
Preferably, in the present invention, the malE gene is inactivated by deletion of the open reading frame base.
By mutating lysC gene encoding aspartokinase so that its initiation codon is mutated from GTG to ATG, its encoded protein carries T311I mutation, and making lysC gene be transcribed by Psod, the enhancement and regulation of aspartokinase expression are finally achieved. The nucleotide sequence of Psod is shown as SEQ ID NO. 37.
The gene hom coding the homoserine dehydrogenase is mutated, so that the coding protein carries G378E mutation, and the hom gene is transcribed by PcspB, so that the deregulation and the expression enhancement of the homoserine dehydrogenase are finally realized. The nucleotide sequence of PcspB is shown as SEQ ID NO. 38.
Enhancement of pyruvate carboxylase expression is finally achieved by mutating the gene pyc encoding pyruvate carboxylase so that its encoded protein carries the P458S mutation and allowing the pyc gene to be transcribed by Psod. The nucleotide sequence of Psod is shown as SEQ ID NO. 37.
By mutating the gene ppc encoding phosphoenolpyruvate carboxylase so that the encoded protein carries the D299N mutation, and allowing the ppc gene to be mutated from P tuf And (3) starting transcription, and finally realizing the expression enhancement of the phosphoenolpyruvate carboxylase. P (P) tuf The nucleotide sequence of (2) is shown as SEQ ID NO. 39.
The invention has the advantages that:
the invention improves the threonine yield of the strain by inactivating the malic enzyme, and can be applied to threonine production. Further, the combination of enhanced expression of one or more of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase or phosphoenolpyruvate carboxylase can increase threonine production by 33.9% compared to the threonine production before modification, and provides a new way for improving threonine production.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The protein and the coding gene thereof related to the invention are as follows:
malic enzyme, coding gene name malE, NCBI accession number: cgl3007, ncgl2904, cg3335;
aspartokinase, coding gene name lysC, NCBI accession number: cgl0251, NCgl0247, cg0306;
homoserine dehydrogenase, coding gene name hom, NCBI accession number: cgl1183, NCgl1136, cg1337;
pyruvate carboxylase, coding gene name pyc, NCBI accession number: cgl0689, NCgl0659, cg0791;
phosphoenolpyruvate carboxylase, coding gene name ppc, NCBI accession number: cgl1585, NCgl1523, cg1787.
According to the invention, firstly, the malic enzyme is inactivated on wild fungus ATCC13032, the obtained modified fungus SMCT336 produces threonine, the threonine yield is 0.2g/L, but due to the strict metabolic regulation in bacteria, the aspartokinase and homoserine dehydrogenase in the threonine synthesis path are strictly regulated by intracellular threonine concentration. Therefore, the invention further opens the threonine synthesis path, mainly comprising demodulation control and expression enhancement of aspartokinase and homoserine dehydrogenase, and the obtained modified bacterium SMCT337 has preliminary threonine synthesis capacity and threonine yield of 2.4g/L.
On this basis, the ability of the strain SMCT338 to produce threonine was increased from 2.4g/L to 3.2g/L by inactivating the malic enzyme.
The invention further researches the threonine production capacity of a series of strains SMCT340, SMCT342 and SMCT344 obtained by carrying out malE inactivation on the strain of SMCT337 for further strengthening and expressing pyruvate carboxylase and at least one enzyme in phosphoenolpyruvate carboxylase, and discovers that the threonine production capacity is improved by 31.4%, 29.7% and 33.9% respectively, and the malic enzyme inactivation is verified to improve the threonine production capacity.
The expression enhancement in the transformation process comprises means such as promoter replacement, ribosome binding site change, copy number increase, plasmid overexpression and the like, and all the means are well known to researchers in the field. The above means are not exhaustive by way of example, and therefore the examples of the present invention will be described with promoter enhancement as representative. The expression attenuation means including promoter substitution, ribosome binding site change, initiation codon substitution, open reading frame base deletion and the like are not intended to be exhaustive, and thus the gene inactivation by the open reading frame base deletion means is represented by the examples of the present invention.
EXAMPLE 1 construction of plasmid for genome engineering of Strain
1) Aspartokinase expression enhancing plasmid pK18mobsacB-P sod -lysC g1a-T311I
Using ATCC13032 genome as a template, performing PCR amplification by using a P21/P22 primer pair to obtain an upstream homology arm up, performing PCR amplification by using a P23/P24 primer pair to obtain a promoter fragment Psod, and performing PCR amplification by using a P25/P26 primer pair to obtain lysC g1a-T311I And (3) carrying out PCR amplification by using a P27/P28 primer pair to obtain a downstream homologous arm dn. And carrying out fusion PCR by taking the P21/P24 primer pair and up and Psod as templates to obtain a fragment up-Psod. With the P21/P28 primer pair with up-Psod, lysC g1a-T311I Fusion PCR is carried out by taking dn as a template to obtain a full-length fragment up-Psod-lysC g1a-T311I Dn. pK18mobsacB was digested with BamHI/HindIII. Assembling the two by using a seamless cloning kit, and transforming a Trans 1T 1 competent cell to obtain a recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I 。
2) Homoserine dehydrogenase expression enhancing plasmid pK18mobsacB-P cspB -hom G378E
PCR amplification was performed with the ATCC13032 genome as a template, the P29/P30 primer pair to obtain an upstream homology arm up, the ATCC14067 genome as a template, the P31/P32 primer pair to obtain a promoter fragment PcspB, and the ATCC13032 genome as a template, the P33/P34 primer pair to obtain hom G378E And (3) carrying out PCR amplification by using a P35/P36 primer pair to obtain a downstream homologous arm dn. Fusion PCR is carried out by taking the P29/P32 primer pair and up and PcspB as templates, so as to obtain fragment up-PcspB. With P29/P36 primer pair in the form of up-PcspB, hom G378E Fusion PCR is carried out by taking dn as a template to obtain a full-length fragment up-PcspB-hom G378E Dn. pK18mobsacB was digested with BamHI/HindIII. Assembling the two by using a seamless cloning kit, and transforming a Trans 1T 1 competent cell to obtain a recombinant plasmid pK18mobsacB-P cspB -hom G378E 。
3) Pyruvate carboxylase expression enhancing plasmid pK18mobsacB-Psod-pyc P458S
Using ATCC13032 genome as a template, performing PCR amplification by using a P13/P14 primer pair to obtain an upstream homology arm up, performing PCR amplification by using a P15/P16 primer pair to obtain a promoter fragment Psod, and performing PCR amplification by using a P17/P18 primer pair to obtain pyc P458S By using a P19/P20 primer pairThe downstream homology arm dn is obtained by PCR amplification. And carrying out fusion PCR by taking the P13/P16 primer pair and up and Psod as templates to obtain a fragment up-Psod. With P13/P20 primer pair with up-Psod, pyc P458S Fusion PCR was performed using dn as a template to obtain the full-length fragment up-Psod-pyc P458S Dn. pK18mobsacB was digested with BamHI/HindIII. The two are assembled by a seamless cloning kit, and Trans 1T 1 competent cells are transformed to obtain a recombinant plasmid pK18mobsacB-Psod-pyc P458S 。
4) Phosphoenolpyruvate carboxylase expression enhancing plasmid pK18mobsacB-Ptuf-ppc D299N
Using ATCC13032 genome as a template, performing PCR amplification by using a P53/P54 primer pair to obtain an upstream homology arm up, performing PCR amplification by using a P55/P56 primer pair to obtain a promoter fragment Ptuf, and performing PCR amplification by using a P57/P58 primer pair to obtain ppc D299N And (3) carrying out PCR amplification by using a P59/P60 primer pair to obtain a downstream homologous arm dn. And (3) performing fusion PCR by using the P53/P56 primer pair and using up and Ptuf as templates to obtain fragments up-Ptuf. With P53/P60 primer pair with up-Ptuf, ppc D299N Fusion PCR is carried out by taking dn as a template to obtain a full-length fragment up-Ptuf-ppc D299N Dn. pK18mobsacB was digested with BamHI/HindIII. Assembling the two with a seamless cloning kit, and transforming a Trans 1T 1 competent cell to obtain a recombinant plasmid pK18mobsacB-Ptuf-ppc D299N 。
5) Malatase inactivating plasmid pK18 mobsacB-. DELTA.malE
The ATCC13032 genome is used as a template, a P181-malE-up-1F/P182-malE-up-1R primer pair is used for PCR amplification to obtain an upstream homology arm up, and a P183-malE-dn-2F/P184-malE-dn-2R primer pair is used for PCR amplification to obtain a downstream homology arm dn. Fusion PCR is carried out by taking up and dn as templates by using a P181-malE-up-1F/P184-malE-dn-2R primer pair to obtain full-length fragments up-dn. pK18mobsacB was digested with BamHI/HindIII. The two are assembled by a seamless cloning kit, and Trans 1T 1 competent cells are transformed to obtain a recombinant plasmid pK18 mobsacB-delta malE.
The primers used in the plasmid construction procedure are shown in Table 1 below:
TABLE 1
EXAMPLE 2 construction of genome-engineered Strain
1) Construction of aspartokinase and homoserine dehydrogenase expression-enhanced Strain
ATCC13032 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I The competent cells were transformed by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target mutant strain is obtained by PCR amplification of the target sequence and nucleotide sequencing analysis. In this strain, the lysC gene was mutated, its initiation codon was mutated from GTG to ATG, the 311 st amino acid sequence encoded by it was mutated from threonine to isoleucine, and the promoter of the lysC gene was replaced with a strong promoter Psod.
Further, the strain was transformed in the above manner, and the homoserine dehydrogenase expression was enhanced (pK 18mobsacB-P was transformed cspB -hom G378E The above modified strain was introduced), and the obtained modified strain was designated as SMCT337. The strain construction method is the same as above. In the strain, the hom gene is further mutated, the corresponding amino acid mutation site is G378E, and the promoter of the hom gene is replaced by a strong promoter PcspB.
2) Construction of pyruvate carboxylase expression-enhancing Strain
Strain construction method referring to 1) above, pyruvate carboxylase expression enhancement was performed using SMCT337 as a starting strain (pK 18mobsacB-Psod-pyc was modified P458S SMCT 337) was introduced, and the resulting engineered strain was designated SMCT339. 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 with a strong promoter P sod 。
3) Construction of phosphoenolpyruvate carboxylase expression-enhancing Strain
Method for constructing Strain referring to 1) above, phosphoenolpyruvate carboxylase expression enhancement modification was performed using SMCT337 and SMCT339 as starting strains (pK 18 mobsacB-Ptuf-ppc) D299N SMCT337 and SMCT339 were introduced, respectively), and the obtained engineered strains were designated as SMCT341 and SMCT343. In the strain, the ppc gene is further mutated, the corresponding amino acid mutation site is D299N, and the promoter of the ppc gene is replaced by a strong promoter P tuf 。
4) Construction of Maltase inactivated expression Strain
Method for constructing strains referring to 1) above, malic enzyme inactivation was modified (pK 18 mobsacB-. DELTA.malE was introduced into ATCC13032, SMCT337, SMCT339, SMCT341 and SMCT343, respectively) using ATCC13032, SMCT337, SMCT339, SMCT341 and SMCT343 as starting strains, and the modified strains obtained were designated as SMCT336, SMCT338, SMCT340, SMCT342 and SMCT344. The malE gene in this strain is deleted in open reading frame base, resulting in its inactivation.
The strains obtained are shown in Table 2.
TABLE 2
Strain name Genotype of the type
SMCT336 ATCC13032,ΔmalE
SMCT337 ATCC13032,Psod-lysC g1a-T311I ，PcspB-hom G378E
SMCT338 ATCC13032,Psod-lysC g1a-T311I ，PcspB-hom G378E ，ΔmalE
SMCT339 ATCC13032,Psod-lysC g1a-T311I ，PcspB-hom G378E ，Psod-pyc P458S
SMCT340 ATCC13032,Psod-lysC g1a-T311I ，PcspB-hom G378E ，Psod-pyc P458S ，ΔmalE
SMCT341 ATCC13032,Psod-lysC g1a-T311I ，PcspB-hom G378E ，Ptuf-ppc D299N
SMCT342 ATCC13032,Psod-lysC g1a-T311I ，PcspB-hom G378E ，Ptuf-ppc D299N ，ΔmalE
SMCT343 ATCC13032,Psod-lysC g1a-T311I ，PcspB-hom G378E ，Psod-pyc P458S ，Ptuf-ppc D299N
SMCT344 ATCC13032,Psod-lysC g1a-T311I ，PcspB-hom G378E ，Psod-pyc P458S ，Ptuf-ppc D299N ，ΔmalE
EXAMPLE 3 construction of strains shake flask verification
1. Culture medium
Seed activation medium: BHI 3.7%, agar 2%, pH 7.0.
Seed culture medium: 5g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 16g/L ammonium sulfate, 8g/L urea, 10.4g/L potassium dihydrogen phosphate, 21.4g/L dipotassium hydrogen phosphate, 5mg/L biotin and 3g/L magnesium sulfate. Glucose 50g/L, pH 7.2.
Fermentation medium: corn steep liquor 50mL/L, glucose 30g/L, ammonium sulfate 4g/L, MOPS 30g/L, monopotassium phosphate 10g/L, urea 20g/L, biotin 10mg/L, magnesium sulfate 6g/L, ferrous sulfate 1g/L, VB1 & HCl40mg/L, calcium pantothenate 50mg/L, nicotinamide 40mg/L, manganese sulfate 1g/L, zinc sulfate 20mg/L, copper sulfate 20mg/L, and pH 7.2.
2. Engineering bacterium shake flask fermentation production of L-threonine
(1) Seed culture: ATCC13032, SMCT336, SMCT337, SMCT338, SMCT339, SMCT340, SMCT341, SMCT342, SMCT343, SMCT344 seed 1 was picked and inoculated into a 500mL Erlenmeyer flask containing 20mL seed medium, and cultured at 30℃under shaking at 220r/min for 16h.
(2) Fermentation culture: 2mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 20mL of the fermentation medium, and cultured at 33℃under 220r/min with shaking for 24 hours.
(3) 1mL of the fermentation broth was centrifuged (12000 rpm,2 min), and the supernatant was collected, and the L-threonine in the fermentation broths of the engineering bacteria and the control bacteria was detected by HPLC, and the concentrations thereof were shown in Table 3 below.
TABLE 3 comparison of threonine-producing Capacity of Corynebacterium glutamicum
As can be seen from the table, the threonine yield of the strain after the malic enzyme is inactivated is improved, wherein the acid production of the SMCT344 is improved from 5.6g/L to 7.5g/L before and after the malic enzyme is inactivated, and the acid production is improved by 33.9 percent relatively; indicating that the inactivation of the malic enzyme can obviously improve the threonine producing capacity of the strain after the threonine end synthesis path is opened; in addition, in a series of strains in which the malate enzyme is inactivated, when the threonine synthesis pathway, the expression intensities of the pyruvate carboxylase and the phosphoenolpyruvate carboxylase are increased, the threonine yield is further improved, which means that the combination of the malate enzyme inactivation and the enhancement of the expression of the above enzymes is advantageous for threonine production.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (9)

1. A modified coryneform microorganism, characterized in that the activity of malic enzyme is reduced or lost compared to an unmodified microorganism, and the microorganism has an enhanced threonine-producing ability compared to an unmodified microorganism.
2. The microorganism of claim 1, wherein the decrease or loss of the activity of the malic enzyme in the microorganism is achieved by decreasing expression of a gene encoding the malic enzyme or knocking out an endogenous gene encoding the malic enzyme.
3. The microorganism of claim 2, wherein the expression of the gene encoding the malic enzyme is reduced or the endogenous gene encoding the malic enzyme is knocked out by mutagenesis, site-directed mutagenesis or homologous recombination.
4. The microorganism of claim 1, wherein the microorganism has an increased activity of an enzyme associated with an in vivo threonine synthesis and/or pre-supply response pathway as compared to an unmodified microorganism;
wherein the enzyme associated with the threonine synthesis and/or precursor supply pathway is selected from at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxylase.
5. The microorganism according to claim 4, wherein the microorganism is any one of the following (1) to (4):
(1) a microorganism having reduced or lost malate activity and enhanced activity of an aspartate kinase and/or a homoserine dehydrogenase;
(2) a microorganism having reduced or lost malate activity and enhanced aspartokinase, homoserine dehydrogenase and/or pyruvate carboxylase activity;
(3) a microorganism having reduced or lost malate activity and enhanced aspartokinase, homoserine dehydrogenase and/or phosphoenolpyruvate carboxylase activity;
(4) a microorganism having reduced or lost malate enzyme activity and enhanced aspartokinase, homoserine dehydrogenase, pyruvate carboxylase and/or phosphoenolpyruvate carboxylase activity.
6. The microorganism according to claim 4, wherein the enhancement of the activity of an enzyme involved in threonine synthesis and/or precursor supply pathway in the microorganism is achieved by a compound selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by altering the nucleotide sequence encoding the enzyme gene.
7. A microorganism according to any of claims 1 to 5, characterized in that the microorganism is corynebacterium glutamicum (Corynebacterium glutamicum).
8. A method for constructing a threonine-producing strain, the method comprising:
A. weakening a gene encoding malic enzyme in coryneform bacteria having amino acid production ability to obtain a gene-weakened strain; the attenuation includes knocking out or reducing expression of a gene encoding a malic enzyme; and/or
B. Enhancing the enzyme related to threonine synthesis and/or precursor supply paths in the gene-attenuated strain of the step A to obtain an enzyme activity-enhanced strain;
the enhanced pathway is selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhancement by altering the nucleotide sequence encoding the enzyme gene;
wherein the enzyme associated with the threonine synthesis and/or precursor supply pathway is selected from at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxylase.
9. A method for producing threonine, characterized in that the method comprises the steps of:
a) Culturing the microorganism of any one of claims 1-7 to obtain a culture of the microorganism;
b) Collecting the threonine produced from the culture obtained in step a).
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