阅读说明：本技术 一种spoT基因改造的重组菌株及其构建方法与应用 (Recombinant strain modified by spoT gene and construction method and application thereof ) 是由 杨立鹏 魏爱英 孟刚 苏厚波 赵春光 马风勇 贾慧萍 于 2019-08-28 设计创作，主要内容包括：本发明公开了一种多核苷酸序列以及包括该多核苷酸序列的产重组菌株,是对大肠杆菌中的spoT基因进行点突变形成,改造后的基因序列如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 polynucleotide sequence and a recombinant strain comprising the polynucleotide sequence, which are formed by point mutation of spoT gene in escherichia coli, wherein the modified gene sequence 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 nucleotide sequence which comprises a nucleotide sequence formed by mutation of 520 th base of a spoT gene coding sequence shown in SEQ ID NO. 1;

preferably, the mutation is that the 520 th base in SEQ ID NO. 1 is mutated from guanine (G) to thymine (T); the nucleotide sequence after mutation is shown as SEQ ID NO. 2.

2. A recombinant protein encoded by the nucleotide sequence of claim 1; for example, the 174 th glycine of the amino acid sequence shown in SEQ ID NO. 3 is substituted by cysteine.

3. The recombinant protein according to claim 2, which comprises the amino acid sequence shown as SEQ ID NO 4.

4. A recombinant vector comprising the nucleotide sequence of claim 1.

5. The recombinant vector according to claim 4, which is constructed by introducing the nucleotide sequence into a plasmid.

6. A recombinant strain comprising the nucleotide sequence of claim 1;

preferably, the recombinant strain is formed by introducing the recombinant vector of claim 4 into a host strain; the host strain is selected from escherichia coli; for example, the host strain is e.coli K12(W3110) strain, e.coli CGMCC 7.232 strain.

7. The method of constructing a recombinant strain according to claim 6, comprising the steps of:

(1) modifying the nucleotide sequence of the open reading frame region of the wild type spoT gene shown as SEQ ID NO. 1 to make the 520 th base of the wild type spoT gene generate mutation so as to obtain the nucleotide sequence shown as SEQ ID NO. 2;

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

(3) and (3) introducing the recombinant vector into a host strain to obtain the recombinant strain.

8. The construction method according to claim 7, wherein the step (1) comprises synthesizing two pairs of primers for amplifying the fragment of the coding region of the spoT gene based on the coding sequence of the spoT gene, and introducing a point mutation into the coding region (SEQ ID NO:1) of the wild-type spoT gene by a PCR site-directed mutagenesis method to obtain a nucleotide sequence (SEQ ID NO:2) of the coding region of the spoT gene with the point mutation, wherein the nucleotide sequence is marked as spoT^(G520T)；

Preferably, the step (1) comprises: e.coli K12 as a template, and primers P1 and P2, P3 and P4 respectively for PCR amplification to obtain two DNA fragments spoT separated from the spoT gene coding region containing point mutation^(G520T)Up and spoT^(G520T)A Down fragment, using the DNA fragment as a template, and using P1 and P4 as primers, and performing overlapping PCR amplification to obtain spoT^(G520T)-Up-Down fragment, said primers P1-P4 being: p1: 5, P2: 6, P3: 7, P4: 8 in SEQ ID NO; preferably, the spoT^(G520T)Up and spoT^(G520T)DNA fragment sizes of-Down were 620bp and 880bp, respectively.

9. The construction method according to claim 7 or 8, wherein the step (2) comprises subjecting the spoT to^(G520T)the-Up-Down fragment and pKOV plasmid were digested with BamH I/NotI, respectively, and the digested spoT was digested with^(G520T)the-Up-Down fragment and the plasmid were separated, purified and ligated by agarose gel electrophoresis to obtain a recombinant vector.

10. The nucleotide sequence, recombinant protein, recombinant vector and recombinant strain of claims 1-6, for use in the preparation of L-threonine, preferably the use comprises fermentation of the recombinant strain to produce L-threonine.

Technical Field

The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to a recombinant strain modified by spoT gene and 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. The L-threonine has the effects of recovering human fatigue and promoting growth and development, and is mainly used in the aspects of medicines, chemical reagents, food enhancers, feed additives and the like. For a long time, the demand of domestic and foreign markets for L-threonine is continuously and stably increased, and particularly, the dosage of L-threonine in the aspects of chemistry, biochemistry, food additives, feed additives and the like is rapidly increased, so that the huge demand of the market causes many domestic enterprises to invest in force to develop threonine products.

The method for producing L-threonine by microbial fermentation is a main industrial production method at present, and has the advantages of low production cost, high production intensity and little environmental pollution. 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. However, L-threonine-producing bacteria also produce metabolic byproducts such as acetic acid, lactic acid, alanine, and aspartic acid while producing L-threonine by fermentation, and thus, the growth of the bacteria and the yield of L-threonine are affected to some extent. There is therefore still a need to develop strains which produce L-threonine more economically with high yields.

Disclosure of Invention

The invention provides a nucleotide sequence which comprises a nucleotide sequence formed by mutation of 520 th base of spoT gene coding sequence shown in SEQ ID NO. 1.

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 520 th base in SEQ ID NO. 1 is mutated from guanine (G) to thymine (T); specifically, the nucleotide sequence is shown as SEQ ID NO. 2.

The present invention provides a recombinant protein encoded by the nucleotide sequence as described above.

The recombinant protein comprises an amino acid sequence shown as SEQ ID NO. 4; specifically, the recombinant protein comprises an amino acid sequence shown in SEQ ID NO. 3, wherein the 174 th glycine is replaced by cysteine.

The present invention provides a recombinant vector comprising the above-described nucleotide sequence or recombinant protein.

The recombinant vector is constructed by introducing the nucleotide sequence into a plasmid; as one embodiment, the plasmid is a pKOV plasmid. Specifically, the nucleotide 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.

The invention further provides a recombinant strain, which contains the spoT gene coding nucleotide sequence with point mutation of the coding sequence, for example, 520 th base of the spoT gene coding nucleotide sequence shown in SEQ ID NO. 1 has point mutation.

According to the recombinant strain, the 520 th base in the SEQ ID NO. 1 sequence is mutated from guanine (G) to thymine (T).

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 is formed by introducing the recombinant vector into a host strain for recombination; the host strain is not particularly limited and may be selected from L-threonine producing strains known in the art that retain the spoT gene, for example, from Escherichia coli. As an embodiment of the present invention, the host strain is e.coli K12(W3110) strain, e.coli cgmcc 7.232 strain.

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

The recombinant strain according to the invention may or may not further comprise other modifications.

The invention provides a construction method of a recombinant strain, which comprises the following steps:

the nucleotide sequence of the spoT gene coding region shown as SEQ ID NO. 1 was modified to mutate the 520 th base to obtain a recombinant strain containing the mutated spoT coding gene.

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 520 th base in SEQ ID NO. 1 is mutated from guanine (G) to thymine (T); specifically, the nucleotide sequence obtained after mutation is shown as SEQ ID NO. 2.

Further, the construction method comprises the following steps:

(1) modifying the nucleotide sequence of the open reading frame region of the wild type spoT gene shown as SEQ ID NO. 1 to make the 520 th base of the wild type spoT gene generate mutation so as to obtain a mutated nucleotide sequence;

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

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

According to the construction method of the present invention, the step (1) includes: constructing the spoT gene coding region with point mutation, namely synthesizing two pairs of primers for amplifying spoT gene coding region fragments according to the spoT gene coding sequence, introducing point mutation in the wild-type spoT gene coding region (SEQ ID NO:1) by a PCR (polymerase chain reaction) site-directed mutagenesis method to obtain the spoT gene coding region nucleotide sequence (SEQ ID NO:2) with point mutation, which is marked as spoT^(G520T)。

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

P1:5'CGGGATCCGAACAGCAAGAGCAGGAAGC 3' (underlined is the restriction site BamH I) (SEQ ID NO:5)

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

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

P4:5'AAGGAAAAAAGCGGCCGCACGACAAAGTTCAGCCAAGC 3' (underlined is restriction enzyme 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, primers P1 and P2, P3 and P4 are respectively used for PCR amplification to obtain two DNA fragments (spoT) with the sizes of 620bp and 880bp separated from the spoT gene coding region containing point mutation^(G520T)Up and spoT^(G520T)-Down fragments). 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 using P1 and P4 as primers to obtain spoT^(G520T)-Up-Down fragment.

In one embodiment of the invention, the spoT is^(G520T)The nucleotide sequence size of the Up-Down fragment is 1500 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, the step (2) comprises constructing a recombinant vector by introducing the spoT into a cell^(G520T)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 spoT was cleaved^(G520T)Separating, purifying and connecting the-Up-Down fragment and the pKOV plasmid through agarose gel electrophoresis to obtain a recombinant vector pKOV-spoT^(G520T)。

According to the construction method of the present invention, the step (3) comprises construction of a recombinant strain: the recombinant vector pKOV-spoT is used^(G520T)And (4) introducing the strain into a host strain to obtain a recombinant strain.

In one embodiment of the present invention, the introduction of the step (3) is an electrical conversion method.

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

The invention also provides a recombinant strain obtained by the construction method.

The present invention provides the use of the recombinant strain as described above for the preparation of L-threonine.

The application of the nucleotide sequence, the recombinant protein, the recombinant vector and the recombinant strain in the preparation of the 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 spoT gene coding sequence in the Escherichia coli producing L-threonine, and compared with a wild strain without mutation, the obtained strain is favorable for producing high-concentration L-threonine and has good strain stability, and the recombinant strain can further reduce the production cost as an L-threonine producing 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.