阅读说明：本技术 一种deoB基因改造的重组菌株及其构建方法与应用 (Recombinant strain modified by deoB gene and construction method and application thereof ) 是由 贾慧萍 孟刚 魏爱英 赵春光 周晓群 马风勇 郭小炜 田斌 于 2019-09-27 设计创作，主要内容包括：本发明公开了一种deoB基因改造的重组菌株及其构建方法与应用,是对大肠杆菌中的deoB基因进行点突变形成,突变后的deoB基因序列如SEQ ID NO:2所示。该重组菌株与未突变的野生型菌株相比,有利于生产高浓度的L-苏氨酸,且菌株稳定性好,作为L-苏氨酸生产菌株能够进一步降低生产成本。(The invention discloses a recombinant strain modified by deoB gene, a construction method and application thereof, which is formed by point mutation of deoB gene in escherichia coli, wherein the sequence of the mutated deoB gene is shown as SEQ ID NO. 2. Compared with a wild strain without mutation, the recombinant strain is favorable for producing high-concentration L-threonine, has good strain stability, and can further reduce the production cost when being used as an L-threonine producing strain.)

1. A polynucleotide sequence is characterized in that the polynucleotide sequence comprises a sequence formed by mutating 1049 th base of a coding sequence of a wild-type deoB gene shown in SEQ ID NO. 1;

preferably, the mutation is that the 1049 th base in SEQ ID NO. 1 is mutated from guanine (G) to adenine (A), and the polynucleotide sequence is shown in SEQ ID NO. 2.

2. A recombinant protein encoded by the polynucleotide sequence of claim 1.

Preferably, the recombinant protein is encoded by the polynucleotide sequence shown in SEQ ID NO. 2.

Preferably, the amino acid sequence is shown as SEQ ID NO. 4.

3. A recombinant vector comprising the polynucleotide sequence of claim 1 or the recombinant protein of claim 2.

4. A recombinant strain comprising a nucleotide sequence encoding a deoB gene having a point mutation in its coding sequence;

preferably, the recombinant strain contains the recombinant vector of claim 3.

5. The recombinant strain of claim 4, wherein the recombinant vector is introduced into a host strain and recombined.

6. The recombinant strain according to claim 5, wherein the host strain is E.coli K12(W3110) strain, E.coli CGMCC7.232 strain.

7. A method of constructing a recombinant strain according to any one of claims 4 to 6, comprising the steps of:

modifying the nucleotide sequence of the open reading frame region of the wild-type deoB gene as shown in SEQ ID NO. 1 to make the 1049 th base of the wild-type deoB gene mutated to obtain an L-threonine-producing recombinant strain containing the mutated deoB coding gene; exemplarily, the mutation is that the 1049 th base in SEQ ID NO. 1 is mutated from guanine (G) to adenine (A); the mutated polynucleotide sequence is shown in SEQ ID NO. 2.

8. The method of constructing a recombinant strain according to claim 7, comprising the steps of:

(1) modifying the nucleotide sequence of the open reading frame region of the wild-type deoB gene as shown in SEQ ID NO. 1 to make the 1049 th base of the wild-type deoB gene mutated to obtain a polynucleotide sequence of the open reading frame region of the mutated deoB gene;

(2) connecting the mutated polynucleotide sequence with a plasmid to construct a recombinant vector;

(3) and (3) introducing the recombinant vector into a host strain to obtain the L-threonine-producing recombinant strain containing the point mutation.

9. The method of constructing a recombinant strain according to claim 8, wherein the step (1) comprises: constructing a deoB gene coding region with point mutation, namely synthesizing two pairs of primers for amplifying deoB gene coding region segments according to the deoB gene coding sequence, introducing point mutation in a wild-type deoB gene coding region SEQ ID NO:1 by a PCR (polymerase chain reaction) site-specific mutagenesis method to obtain a nucleotide sequence SEQ ID NO:2 of the deoB gene coding region with point mutation, which is marked as deoB^(G1049A)；

Preferably, in the step (1), the primers are:

P1:5'CGGGATCCATGGACGGCAACGCTGAAG 3'

P2:5'GATCGTAACCGTGGTCAG 3'

P3:5'CTGACCACGGTTACGATC 3'

P4:5'AAGGAAAAAAGCGGCCGCGCTCGTGAGTGCGGATGT 3'。

10. use of the recombinant strain according to any one of claims 4 to 6 for the fermentative preparation of L-threonine or for increasing the fermentative content of L-threonine.

Technical Field

The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to a deoB gene modified recombinant strain, a construction method and application thereof.

Background

L-threonine is one of the eight essential amino acids, and is an amino acid that cannot be synthesized by humans and animals themselves. L-threonine can strengthen the absorption of grains, regulate the metabolism balance in vivo and promote the growth and development of organisms, and is widely applied to the feed, medicine and food industries.

At present, the production of L-threonine mainly comprises a chemical synthesis method, a protein hydrolysis method and a microbial fermentation method, wherein the microbial fermentation method has low production cost, high production intensity and small environmental pollution, thereby becoming the most widely applied method for industrial production of L-threonine at present. Various bacteria can be used for the microbial fermentation production of L-threonine, such as mutant strains obtained by wild-type induction of Escherichia coli, Corynebacterium, Serratia, and the like, as production strains. Specific examples include amino acid analogue resistant mutants or various auxotrophs such as methionine, threonine, isoleucine and the like. However, in the conventional mutation breeding, the strain grows slowly and generates more byproducts due to random mutation, so that a high-yield strain is not easy to obtain. Therefore, the construction of recombinant Escherichia coli by metabolic engineering is an effective way to produce L-threonine.

At present, the overexpression or attenuation of key enzyme genes in an amino acid synthesis path and a competition path mediated by expression plasmids is a main means for carrying out genetic modification on Escherichia coli. There is still a need to develop a method for producing L-threonine more economically with high yield.

Disclosure of Invention

In a first aspect, the present invention provides a polynucleotide sequence comprising a sequence of the wild-type deoB gene coding sequence of SEQ ID NO. 1 in which the 1049 th base is mutated.

According to the present invention, the mutation refers to a change in the base/nucleotide at the site, and the mutation method may be at least one selected from the group consisting of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.

According to the invention, the base 1049 in SEQ ID NO. 1 is mutated from guanine (G) to adenine (A); specifically, the polynucleotide sequence is shown as SEQ ID NO. 2.

In a second aspect of the invention, there is provided a recombinant protein encoded by a polynucleotide sequence as described above.

The recombinant protein comprises an amino acid sequence shown as SEQ ID NO. 4.

In a third aspect of the invention, there is provided a recombinant vector comprising the polynucleotide sequence described above.

The recombinant vector is constructed by introducing the polynucleotide sequence into a plasmid; as one embodiment, the plasmid is a pKOV plasmid. Specifically, the polynucleotide sequence and the plasmid may be digested with endonuclease to form complementary cohesive ends, and the two may be ligated to construct a recombinant vector.

In a fourth aspect of the invention, there is provided a recombinant strain comprising a nucleotide sequence encoding a deoB gene in which the coding sequence is point mutated.

A recombinant strain according to the invention comprising a polynucleotide sequence as described in the first aspect.

As one embodiment of the present invention, it contains the nucleotide sequence shown as SEQ ID NO. 2.

As one embodiment of the present invention, it contains the amino acid sequence shown as SEQ ID NO. 4.

The recombinant strain according to the present invention is formed by introducing the recombinant vector according to the third aspect of the present invention into a host strain; the host strain is not particularly limited and may be selected from L-threonine producing strains known in the art that retain the deoB gene, for example, from Escherichia coli. As an embodiment of the present invention, the host strain is e.colik12(W3110) strain, e.coli CGMCC7.232 strain.

The recombinant strain of the invention takes pKOV plasmid as a vector.

The recombinant strain according to the present invention may further comprise other modifications.

In the fifth aspect of the present invention, there is also provided a method for constructing a recombinant strain, comprising the steps of:

the nucleotide sequence of the open reading frame region of the wild-type deoB gene shown as SEQ ID NO. 1 is reformed, and the 1049 th base of the wild-type deoB gene is mutated, so that the L-threonine-producing recombinant strain containing the mutant deoB coding gene is obtained.

According to the construction method of the invention, the modification comprises at least one of mutagenesis, PCR site-directed mutagenesis, homologous recombination and the like.

According to the construction method of the invention, the mutation is that the 1049 th base in SEQ ID NO. 1 is mutated from guanine (G) to adenine (A); specifically, the mutated polynucleotide sequence is shown in SEQ ID NO. 2.

Illustratively, the construction method comprises the following steps:

(1) modifying the nucleotide sequence of the open reading frame region of the wild-type deoB gene as shown in SEQ ID NO. 1 to make the 1049 th base of the wild-type deoB gene mutated to obtain a polynucleotide sequence of the open reading frame region of the mutated deoB gene;

(2) connecting the mutated polynucleotide sequence with a plasmid to construct a recombinant vector;

(3) and (3) introducing the recombinant vector into a host strain to obtain the L-threonine-producing recombinant strain containing the point mutation.

According to the construction method of the present invention, the step (1) includes: constructing a deoB gene coding region with point mutation, namely synthesizing two pairs of primers for amplifying a deoB gene coding region fragment according to a deoB gene coding sequence, introducing point mutation in a wild-type deoB gene coding region (SEQ ID NO:1) by a PCR (polymerase chain reaction) site-directed mutagenesis method to obtain a nucleotide sequence (SEQ ID NO:2) of the deoB gene coding region with point mutation, and marking as deoB^(G1049A)。

In one embodiment of the present invention, in the step (1), the primers are:

P1:5'CGGGATCCATGGACGGCAACGCTGAAG 3' (underlined part is the restriction endonuclease cleavage site BamH I) (SEQ ID NO:5)

P2:5'GATCGTAACCGTGGTCAG 3'(SEQ ID NO:6)

P3:5'CTGACCACGGTTACGATC 3'(SEQ ID NO:7)

P4:5'AAGGAAAAAAGCGGCCGCGCTCGTGAGTGCGGATGT 3' (underlined is restriction endonuclease cleavage site Not I) (SEQ ID NO: 8);

in one embodiment of the present invention, the step (1) comprises: e.coli K12 as a template, and primers P1/P2 and P3/P4 respectively, to perform PCR amplification, and obtain two DNA fragments (deoB Up and deoB Down) with sizes of 836bp and 890bp separated from the coding region of deoB gene. Separating and purifying the two DNA fragments by agarose gel electrophoresis, and performing Overlap PCR amplification (Overlap PCR) by using the two DNA fragments as templates and P1 and P4 as primers to obtain deoB^(G1049A)-Up-Down。

In one embodiment of the invention, said deoB^(G1049A)The size of the-Up-Down nucleotide sequence is 1726 bp.

In one embodiment of the invention, the PCR amplification is performed as follows: denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 30s (30 cycles).

In one embodiment of the invention, the overlapping PCR amplification is performed as follows: denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 60s (30 cycles).

According to the construction method of the present invention, said step (2) comprises construction of a recombinant vector by introducing the above deoB^(G1049A)the-Up-Down fragment was separated and purified by agarose gel electrophoresis, and then cleaved with BamH I/Not I in a double-restriction enzyme together with pKOV plasmid, and the cleaved deoB fragment was cleaved with BamH I/Not I in a double-restriction enzyme^(G1049A)Separating, purifying and connecting the-Up-Down fragment and the pKOV plasmid through agarose gel electrophoresis to obtain a recombinant vector pKOV-deoB^(G1049A)。

According to the construction method of the present invention, the step (3) comprises construction of a recombinant strain: the recombinant vector pKOV-deoB^(G1049A)And transforming the host strain to obtain a recombinant strain.

In one embodiment of the present invention, the conversion of step (3) is an electrical conversion process; illustratively, in the step (3), a recombinant vector is introduced into the host strain.

The construction method according to the present invention further comprises the step of screening the recombinant strain; illustratively, screening is performed using chloramphenicol medium.

In a sixth aspect of the present invention, there is also provided a recombinant strain obtained by the construction method as described above. Furthermore, the construction method according to the fifth aspect of the present invention can be used for constructing a recombinant strain according to the fourth aspect.

In a seventh aspect of the present invention, there is provided a use of the recombinant strain according to the fourth or sixth aspect for the production of L-threonine or for increasing the fermentation amount of L-threonine.

The application of the recombinant strain in the preparation of L-threonine comprises the step of fermenting the recombinant strain to prepare the L-threonine.

Advantageous effects

The recombinant strain is obtained by introducing point mutation into the deoB gene coding sequence in the wild corynebacterium glutamicum, and compared with the wild strain without mutation, the obtained strain is favorable for producing high-concentration L-threonine and has good strain stability, and the production cost can be further reduced when the strain is used as an L-threonine production strain.

Detailed Description

The present invention will be described in further detail with reference to examples. However, those skilled in the art will appreciate that the scope of the present invention is not limited to the following examples. In light of the present disclosure, those skilled in the art will recognize that many variations and modifications may be made to the embodiments described above without departing from the spirit and scope of the present invention.