CN118165903A - Genetically engineered bacteria producing L-threonine and its application - Google Patents

Abstract

The invention discloses a genetically engineered bacterium producing L-threonine and its application. The invention strengthens the biotin synthesis of the L-threonine producing strain by strengthening the birA gene. The genetically engineered bacterium producing L-threonine can produce 8.3 g/L of L-threonine and a sugar-acid conversion rate of 27.8% after fermentation culture.

Description

Genetically engineered bacteria producing L-threonine and its application

The present invention is a divisional application of the Chinese patent application with application number CN202111241433.2 and invention name “Genetically engineered bacteria producing L-threonine and construction method and application thereof”.

Technical Field

The present invention relates to the field of biotechnology, in particular to a genetically engineered bacterium producing L-threonine and its application.

Background technique

L-threonine is one of the 8 kinds of amino acids necessary for human and animal growth, and is widely used in feed, food additives and preparation of pharmaceutical auxiliary materials. At present, L-threonine is mainly produced by microbial fermentation, and various bacteria can be used for L-threonine production, such as mutants obtained by wild-type induction of Escherichia coli, Corynebacterium, Serratia, etc. as production strains. Specific examples include mutants resistant to amino acid analogs or various nutritional deficiency types such as methionine, lysine, isoleucine (Japanese Patent Application Publication No. 224684/83; Korean Patent Application Publication No. 8022/87). However, traditional mutagenesis breeding is difficult to obtain high-yield strains due to slow growth of strains and production of more by-products caused by random mutations.

With the increasing global demand for threonine, the construction and transformation of high-yield threonine strains is particularly important. In 2003, CJ Co., Ltd. of South Korea applied for Chinese patent CN03811059.8, using Escherichia coli, by deleting the 39bp sequence from -56 to -18 of the threonine operon sequence, the expression of the key gene thrABC for threonine synthesis was enhanced, and the threonine productivity was increased by 22%. Kwang Ho Lee (Kwang Ho Lee et al., Systems metabolic engineering of Escherichia coli for L-threonine production, Mol Syst Biol. 2007; 3: 149) et al. used the system metabolic engineering strategy to remove product feedback inhibition by mutating the genes thrA and lysC encoding aspartate kinases I and III, removing byproducts glycine and isoleucine by knocking out tdh and weakening ilvA, and providing more precursors for threonine synthesis by inactivating the competitive pathway genes metA and lysA. The TH28C (pBRThrABCR3) strain finally obtained can produce 82.4 g/L of acid in 50 hours of fermentation, and the sugar-acid conversion rate is 39.3%. CN105543156A obtained the MHZ-0213-3 strain by strengthening thrA*BC and knocking out tdh. The strain has a threonine production of 4.2 g/L and a conversion rate of about 8.9%, and has no plasmid burden. CN106635945A obtains the MHZ-0215-2 strain by strengthening the pntAB gene and heterologously introducing the pyc gene in Escherichia coli. The strain has a threonine yield of 12.4 g/L, a conversion rate of about 16.2%, and no plasmid burden.

Summary of the invention

The purpose of the present invention is to provide a novel genetically engineered bacterium for producing L-threonine and its application.

The present invention is conceived as follows: first, a strain capable of producing threonine is constructed, and the expression of enzymes related to the threonine synthesis pathway in Corynebacterium glutamicum (mainly pyruvate carboxylase, aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase) is enhanced, thereby improving the amino acid production capacity of Corynebacterium glutamicum, especially the production capacity of L-threonine; on this basis, the expression of the biotin synthesis birA gene is enhanced to further improve the threonine production capacity.

In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a genetically engineered bacterium producing L-threonine, wherein the genetically engineered bacterium producing L-threonine is a genetically engineered bacterium obtained by strengthening the biotin synthesis pathway of the strain through genetic engineering means.

The strain is a strain capable of producing threonine, preferably a bacterium in the genus Corynebacterium, more preferably Corynebacterium glutamicum.

The genetically engineered bacteria are obtained by enhancing the gene birA (NCBI No.: cg0814, Cgl0709, NCgl067) in Corynebacterium glutamicum.

The enhanced approach can be selected from at least one of the following 1) to 3):

1) Enhanced by operably linking a strong promoter to a biotin synthesis gene;

2) Enhanced by increasing the copy number of biotin synthesis genes on chromosomes;

3) Enhanced by increasing the strength of the RBS sequence of the biotin synthesis gene promoter on the chromosome.

Considering that the enhancement and weakening of gene expression cannot be exhaustively listed through examples, the present invention only provides illustrative examples, and technicians can make adaptive adjustments according to specific circumstances during actual operations. Therefore, the enhancement and weakening referred to in the present invention include but are not limited to the contents disclosed in the embodiments.

The strong promoter can be selected from Ptac, Plac, Ptrp, Ptrc, Psod and the like.

Furthermore, the Corynebacterium glutamicum is a model strain in which the expression of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine synthase, pyruvate carboxylase, etc. is enhanced.

In a second aspect, the present invention provides a method for constructing a genetically engineered bacterium producing L-threonine, which utilizes genetic engineering means to operably connect the strong promoter Psod with birA in Corynebacterium glutamicum to achieve the purpose of enhancing the expression of the birA gene.

In a third aspect, the present invention provides a genetically engineered bacterium producing L-threonine constructed according to the above method.

In a fourth aspect, the present invention provides the use of the genetically engineered bacteria, or the genetically engineered bacteria constructed according to the above method, in the fermentation production of L-threonine.

The present invention only provides some examples of gene expression enhancement in the biotin synthesis pathway. Those skilled in the art may make certain changes to enhance the synthesis pathway, including but not limited to relieving feedback inhibition, promoter enhancement, ribosome binding site enhancement, and copy number increase. All of the above methods can enhance the biotin synthesis pathway and fall within the scope of protection of the present invention.

In addition, the expression of pyruvate carboxylase, aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase in Corynebacterium glutamicum is enhanced, including but not limited to release of feedback inhibition, promoter enhancement, ribosome binding site enhancement, copy number increase, etc.

In addition, the coryneform bacteria that produce threonine are not limited to the coryneform bacteria glutamicum, and the engineering modification thereof may be a combination of the above-mentioned enzymes, namely, pyruvate carboxylase, aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase.

By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

The present invention strengthens the biotin synthesis of the L-threonine production strain by strengthening the expression of the birA gene, increasing the copy number, etc. The constructed L-threonine-producing genetically engineered bacteria can produce up to 8.3 g/L of L-threonine and a sugar-acid conversion rate of 27.8% after fermentation. The present invention is also applicable to increasing the production and sugar-acid conversion rate of aspartic acid family amino acids such as aspartic acid, lysine, homoserine, methionine, isoleucine, etc. In addition, the present invention is also applicable to other enzymes that use biotin as a cofactor to increase the enzyme activity and thus enhance the synthesis path of amino acids.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

The reagents used in the following examples are all commercially available. The high conversion rate threonine production strain Corynebacterium glutamicum provided by the present invention is constructed from a wild strain.

Example 1 Construction of auxiliary plasmids required for genome transformation of Corynebacterium glutamicum

1. Plasmid pK18mobsacB-P _sod -lysC ^V1M-T311I for enhanced expression of aspartate kinase

Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, the upstream homology arm up was obtained by PCR amplification with the primer pair P21/P22, the promoter fragment P _sod was obtained by PCR amplification with the primer pair P23/P24, lysC ^V1M-T311I was obtained by PCR amplification with the primer pair P25/P26, and the downstream homology arm dn was obtained by PCR amplification with the primer pair P27/P28. The fragment up-P _sod was obtained by fusion PCR with the primer pair P21/P24 and up and P _sod as templates. The full-length fragment up-P _sod -lysC ^V1M-T311I -dn was obtained by fusion PCR with the primer pair P21/P28 and up-P _sod , lysC ^{V1M -T311I} , and dn as templates. pK18mobsacB was digested with BamHI/HindIII. The two were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-P _sod -lysC ^V1M-T311I .

2. Plasmid pK18mobsacB-P _sod -asd for enhanced expression of aspartate semialdehyde dehydrogenase

The plasmid construction method was the same as above, and the primers used were: P1, P2, P3, P4, P5, and P6.

3. Plasmid pK18mobsacB-P _cspB -hom ^G378E for enhanced expression of homoserine dehydrogenase

The plasmid construction method was the same as above, and the primers used were: P29, P30, P31, P32, P33, P34, P35, and P36.

4. Plasmid pK18mobsacB-P _cspB -thrB for enhanced expression of homoserine kinase

The plasmid construction method was the same as above, and the primers used were: P7, P8, P9, P10, P11, and P12.

5. Plasmid pK18mobsacB-P _sod -thrC ^V1M for enhanced expression of threonine synthase

The plasmid construction method was the same as above, and the primers used were: P37, P38, P39, P40, P41, and P42.

6. Plasmid pK18mobsacB-P _sod -pyc ^P458S for enhanced expression of pyruvate carboxylase

The plasmid construction method is the same as above, and the primers used are: P13, P14, P15, P16, P17, P18, P19, P207, and the plasmid pK18mobsacB-P _tuf -birA with enhanced biotin synthesis pathway

The plasmid construction procedures were the same as above, and the primers used were: P43, P44, P45, P46, P47, and P48.

The primer sequences used in plasmid construction are shown in Table 1.

Table 1

Example 2 Construction of engineered bacteria of Corynebacterium glutamicum

1. Construction of strains with enhanced expression of aspartate kinase

ATCC 13032 competent cells were prepared according to the classic method of C. glutamicum Handbook, Charter 23. The competent cells were transformed with the recombinant plasmid pK18mobsacB-Psod-lysC ^V1M-T311I by electroporation, and transformants were screened on a selection medium containing 15 mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in a common liquid brain heart infusion medium 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 diluted in a continuous gradient (10 ^-2 to 10 ^-4 ), and the dilution was spread on a common solid brain heart infusion medium containing 10% sucrose and cultured at 33°C for 48 hours. The strain grown on the sucrose medium did not carry the inserted vector sequence in its genome. The target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was named SMCT039.

2. Construction of aspartate semialdehyde dehydrogenase enhanced strain

The strain construction method was the same as above, and aspartate semialdehyde dehydrogenase was enhanced on SMCT039, and the obtained transformed strain was named SMCT040.

3. Construction of homoserine dehydrogenase enhanced strain

The strain construction method was the same as above, and homoserine dehydrogenase was enhanced on SMCT040, and the obtained transformed strain was named SMCT041.

4. Construction of homoserine kinase enhanced strain

The strain construction method was the same as above, and homoserine kinase enhancement was performed on SMCT041, and the obtained transformed strain was named SMCT042.

5. Construction of threonine synthase enhanced strain

The strain construction method was the same as above. The threonine synthase enhanced strain was constructed on SMCT042, and the obtained transformed strain was named SMCT043.

6. Construction of pyruvate carboxylase enhanced strain

The strain construction method was the same as above. The pyruvate carboxylase enhanced strain was constructed on SMCT043, and the obtained transformed strain was named SMCT044.

7. Construction of strains with enhanced biotin synthesis pathway

The strain construction method was the same as above, except that the bifunctional biotin ligase/biotin operon repressor birA promoter was replaced on SMCT044, and the resulting transformed strain was named SMCT045.

The engineered strains are shown in Table 2:

Table 2

Strain name genotype SMCT039 ATCC13032,P _sod -lysC ^V1M-T311I SMCT040 ATCC13032,P _sod -lysC ^V1M-T311I ,P _sod -asd SMCT041 ATCC13032,P _sod -lysC ^V1M-T311I ,P _sod -asd,PcspB-hom ^G378E SMCT042 ATCC13032,P _sod -lysC ^V1M-T311I ,P _sod -asd,P cspB -hom ^G378E ,P _cspB -thrB SMCT043 ATCC13032,P _sod -lysC ^V1M-T311I ,P _sod -asd,PcspB-hom ^G378E ,P _cspB -thrB,P _sod -thrC ^V1M SMCT044 ATCC13032,P _sod -lysC ^V1M-T311I ,P _sod -asd,PcspB-hom ^G378E ,P _cspB -thrB,P _sod -thrC ^V1M ,P _sod -pyc ^P458S SMCT045 ATCC13032,P _sod -lysC ^V1M-T311I ,P _sod -asd,PcspB-hom ^G378E ,P _cspB -thrB,P _sod -thrC ^V1M ,P _sod -pyc ^P458S ,P _tuf -birA

Example 3 Performance verification of L-threonine production by engineered bacteria of Corynebacterium glutamicum

1. Culture medium

Seed activation medium: BHI 3.7%, agar 2%, pH 7.

Seed culture 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, potassium hydrogen phosphate 21.4g/L, biotin 5mg/L, magnesium sulfate 3g/L. Glucose 50g/L, pH 7.2.

Fermentation medium: corn steep liquor 50 mL/L, glucose 45 g/L, ammonium sulfate 4 g/L, MOPS 30 g/L, potassium dihydrogen phosphate 10 g/L, urea 20 g/L, biotin 10 mg/L, magnesium sulfate 6 g/L, ferrous sulfate 1 g/L, VB1·HCl 40 mg/L, calcium pantothenate 50 mg/L, nicotinamide 40 mg/L, manganese sulfate 1 g/L, zinc sulfate 20 mg/L, copper sulfate 20 mg/L, pH 7.2.

2. Production of L-threonine by Shake Flask Fermentation of Engineered Bacteria

(1) Seed culture: Select SMCT021, SMCT023, SMCT031, SMCT033, SMCT034, SMCT035, SMCT036, and SMCT037 slant seeds and inoculate them into a 500-mL Erlenmeyer flask containing 20 mL of seed culture medium. Culture them at 30°C and 220 rpm for 16 h.

(2) Fermentation culture: 2 mL of seed solution was inoculated into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium and cultured at 33 °C and 220 rpm for 24 h.

(3) 1 mL of the fermentation broth was centrifuged (12000 rpm, 2 min), the supernatant was collected, and L-threonine in the fermentation broth of the engineered bacteria and the control bacteria was detected by HPLC. The results are shown in Table 3:

Table 3 Comparison of threonine production capacity of Corynebacterium glutamicum

It can be seen that the threonine production of the strain after the biotin synthesis pathway was strengthened increased from 6.4 g/L to 8.3 g/L, an increase of 30%, indicating that after the terminal threonine synthesis pathway was opened, the improvement of biotin synthesis ability can significantly enhance the strain's ability to produce threonine.

Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (8)

1. The genetically engineered bacterium for producing L-threonine is characterized in that the genetically engineered bacterium for producing L-threonine is obtained by adopting a genetic engineering means and strengthening a biotin synthesis path of a strain;

The strain is a strain having threonine-producing ability, preferably a bacterium of the genus Corynebacterium (Corynebacterium).

2. The genetically engineered bacterium according to claim 1, which is obtained by enhancing a gene birA encoding NCBI reference sequences cg0814, cgl0709, NCgl067 in corynebacterium glutamicum (Corynebacterium glutamicum).

3. The genetically engineered bacterium of claim 2, wherein the pathway of enhancement is selected from at least one of the following 1) to 3):

1) Enhanced by operably linking a strong promoter to a biotin synthesis gene;

2) Enhanced by increasing the copy number of the biotin synthesis genes on the chromosome;

3) Enhanced by increasing the strength of the RBS sequence of the biotin synthesis gene promoter on the chromosome.

4. The genetically engineered bacterium of claim 3, wherein the strong promoter is selected from Ptac, plac, ptrp, ptrc, psod.

5. The genetically engineered bacterium of any one of claims 1-4, wherein the corynebacterium glutamicum is a strain in which the mode strain is enhanced in expression by at least one of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase, threonine synthase, and pyruvate carboxylase.

6. A method for constructing an L-threonine-producing genetically engineered bacterium, characterized in that a strong promoter Psod is operably linked to a gene birA in Corynebacterium glutamicum by genetic engineering means.

7. The L-threonine-producing genetically engineered bacterium constructed according to the method of claim 6.

8. Use of the genetically engineered bacterium of any one of claims 1-5 or claim 7 in the fermentative production of L-threonine.