阅读说明：本技术 一种sdaA基因的启动子核酸序列、含有该核酸序列的重组菌株及其应用 (Promoter nucleic acid sequence of sdaA gene, recombinant strain containing nucleic acid sequence and application of recombinant strain ) 是由 马风勇 魏爱英 孟刚 周晓群 杨立鹏 苏厚波 贾慧萍 郭小炜 于 2019-09-29 设计创作，主要内容包括：本发明提供一种sdaA基因的启动子核酸序列、含有该核酸序列的重组菌株及其应用,该启动子是在sdaA基因的野生型启动子序列中引入点突变获得。本发明的重组菌株用于发酵生产L-赖氨酸,进一步提高了L-赖氨酸的产量,而且与现有其他经改造的产L-赖氨酸菌株的改造位点没有冲突,实现了新的提高L-赖氨酸的发酵量的方式,便于推广应用。(The invention provides a promoter nucleic acid sequence of an sdaA gene, a recombinant strain containing the nucleic acid sequence and application thereof, wherein the promoter is obtained by introducing point mutation into a wild-type promoter sequence of the sdaA gene. The recombinant strain is used for producing L-lysine by fermentation, the yield of the L-lysine is further improved, and the recombinant strain has no conflict with the modification sites of other modified L-lysine-producing strains, so that a novel mode for improving the fermentation amount of the L-lysine is realized, and the popularization and the application are convenient.)

1. The promoter nucleotide sequence is characterized by comprising a sequence with mutation at the-303 bp position of the nucleotide sequence of the wild promoter region shown in SEQ ID NO. 1.

2. The promoter nucleotide sequence of claim 1, wherein the promoter nucleotide sequence is selected from the group consisting of:

(a) as shown in SEQ ID NO. 2; or is

(b) A nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO. 2 and which retains the enhanced activity of the promoter of (a) and the-303 bp position is maintained as cytosine (C).

3. An expression cassette comprising the promoter nucleotide sequence of claim 1 or 2, and a coding sequence operably linked after the promoter nucleotide sequence,

preferably, the coding sequence is the coding sequence of the sda gene.

4. A recombinant vector comprising the promoter nucleotide sequence of claim 1 or 2;

preferably, the recombinant vector comprises a shuttle plasmid; for example, the shuttle plasmid is the pK18mobsacB plasmid.

5. A recombinant strain comprising the promoter nucleotide sequence of claim 1 or 2; or comprising an expression cassette according to claim 3 or a recombinant vector according to claim 4, or the recombinant strain is transformed with an expression cassette according to claim 3 or a recombinant vector according to claim 4.

6. The recombinant strain according to claim 5, which is derived from Corynebacterium or Escherichia coli as host bacterium, preferably Corynebacterium glutamicum.

7. The method of constructing a recombinant strain according to any one of claims 5 to 6,

(1) transforming a wild type promoter region shown as SEQ ID NO.1 to mutate-303 bp nucleotides to obtain a mutant promoter nucleotide sequence;

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

(3) introducing the recombinant vector into a host strain to obtain the L-lysine-producing recombinant strain containing the mutant promoter region;

preferably, in the step (1), the method for mutating is a PCR site-directed mutagenesis method;

preferably, the promoter nucleotide sequence obtained in step (1) is as defined in claim 1 or 2.

8. The construction method according to claim 7, wherein the step (1) comprises synthesizing two pairs of primers P1, P2, P3, P4 for amplifying the wild-type promoter region fragment, and obtaining the promoter nucleotide sequence containing the mutation by PCR site-directed mutagenesis;

preferably, the primers are respectively shown as the following sequences: p1 SEQ ID NO.3, P2 SEQ ID NO.4, P3 SEQ ID NO. 5, P4 SEQ ID NO. 6;

preferably, the PCR site-directed mutagenesis comprises: carrying out PCR amplification by using Corynebacterium glutamicum ATCC13032 as a template and primers P1/P2 and P3/P4 respectively to obtain two DNA fragments with sizes of 660bp and 626bp and separated from a promoter region; the nucleotide sequence of the mutant promoter region was obtained by Overlap PCR amplification (Overlap PCR) using the above two DNA fragments as templates and P1 and P4 as primers.

9. Use of a recombinant strain according to any one of claims 5 to 6 for the production of L-lysine.

10. A method for increasing the fermentation yield of L-lysine or producing L-lysine, which comprises fermenting the recombinant strain of any one of claims 5 to 6.

Technical Field

The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to a promoter nucleic acid sequence of an sdaA gene, a recombinant strain containing the nucleic acid sequence and application of the nucleic acid sequence.

Background

L-lysine is one of the eight major amino acids essential for human and animal life activities. L-lysine has various physiological functions, such as regulating metabolic balance, promoting human development, promoting the absorption of cereal protein and other amino acids by the body, enhancing the immune function and the like, and is widely applied to various aspects such as food additives, animal feeds, drug synthesis and the like.

The production method of L-lysine mainly comprises a protein hydrolysis method, a microbial fermentation method and the like. Among them, the microbial fermentation method has become the most common method for industrially producing L-lysine due to its advantages of easy control of production process, high productivity, etc. The microbial fermentation method is to realize the accumulation of L-lysine in the fermentation liquid by fermenting the microbes capable of metabolizing and synthesizing lysine and utilizing the microbial metabolism.

Microorganisms for producing L-lysine include various species such as coryneform bacteria, Bacillus, Escherichia, and the like. However, wild-type strains have poor L-lysine productivity and a large number of metabolic byproducts, and it is difficult to prepare L-lysine with high purity and high yield. Therefore, it is generally desired to obtain a strain having a high L-lysine yield. At present, methods for obtaining high-yield strains mainly comprise mutation screening breeding or genetic engineering breeding. Mutation breeding means that a strain is induced to generate nonspecific gene site mutation by ultraviolet irradiation or stimulation of other external conditions, and then a high-yield strain is obtained by screening. Genetic engineering is the optimization of selected strains by means of defined genetic modifications, for example by introducing beneficial enzyme genes with increased enzyme activity by increased copy or site-directed mutagenesis, or by knocking out undesirable genes to abolish enzyme activity/expression. Corynebacterium glutamicum (Corynebacterium glutamicum) is the most commonly used L-lysine-producing strain, which synthesizes L-lysine through metabolic processes such as the Diaminopimelic Acid (DAP) pathway. The availability of C.glutamicum with high L-lysine yields is of great importance for L-lysine production.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a promoter nucleotide sequence, a recombinant strain containing the nucleotide sequence, a construction method of the recombinant strain and application of the recombinant strain in fermentation production of L-lysine. The mutant recombinant strain with high L-lysine yield is obtained by performing site-specific mutagenesis on the nucleotide sequence of the promoter region of the sdaA gene, and when the mutant recombinant strain is used for producing L-lysine by fermentation, the L-lysine yield is obviously improved compared with that of a wild promoter region.

The invention is realized by adopting the following technical scheme:

in the first aspect of the invention, a promoter nucleotide sequence is provided, which comprises a nucleotide sequence with mutation at the-303 bp position of the nucleotide sequence of the wild-type promoter region shown in SEQ ID NO. 1. In one embodiment of the present invention, the promoter serves as a promoter of the sdaA gene.

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

According to the invention, the-303 bp position of the nucleotide sequence of the promoter is mutated from adenine (A) to cytosine (C). Specifically, the nucleotide sequence of the promoter is as follows:

(a) a nucleotide sequence shown as SEQ ID NO. 2;

or (b) a nucleotide sequence which has 90% or more identity to the nucleotide sequence shown in SEQ ID NO. 2 and which retains the enhanced activity of the promoter of (a) and the-303 bp position remains cytosine (C).

In a second aspect of the invention, there is provided an expression cassette comprising the promoter described above, comprising said promoter and a coding sequence operably linked thereto. In one embodiment of the invention, the coding sequence is the coding sequence of the sdaA gene.

In a third aspect of the present invention, there is provided a recombinant vector comprising the above-mentioned promoter nucleotide sequence.

According to the present invention, the above-mentioned mutated promoter nucleotide sequence is linked to a shuttle plasmid to construct the recombinant vector; as an embodiment of the present invention, the shuttle plasmid is a pK18mobsacB plasmid.

In a fourth aspect of the present invention, there is provided a recombinant strain comprising the above-described promoter nucleotide sequence.

The recombinant strain comprises a nucleotide sequence shown as SEQ ID NO. 2. The nucleotide sequence shown in SEQ ID NO. 2 is a promoter region of an sdaA gene; further, the nucleotide sequence shown in SEQ ID NO. 2 is connected with the coding sequence of the sdaA gene. In particular, the recombinant strain may comprise or be transformed with an expression cassette according to the second aspect or a recombinant vector according to the third aspect.

According to the recombinant strain, the mutant promoter nucleotide sequence is introduced into a host strain to be recombined; the host strain may be selected from L-lysine producing strains known in the art, for example, at least one selected from Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium glutamicum, Corynebacterium pekinense; corynebacterium glutamicum is preferred. As an embodiment of the present invention, the host strain is YP097158 (accession No. CGMCC No.12856, accession No. 2016, 8/16/2016).

The recombinant strain of the invention takes pK18mobsacB plasmid as a vector.

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

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

the wild type promoter region of the sdaA gene shown in SEQ ID NO.1 is modified to mutate the-303 bp nucleotides thereof, and an L-lysine-producing recombinant strain containing the mutated promoter region is obtained.

According to the invention, the mutation is that the-303 bp position of the nucleotide sequence of the promoter is mutated from adenine (A) to cytosine (C); specifically, the nucleotide sequence of the mutant promoter region is shown as SEQ ID NO. 2.

Further, the construction method comprises the following steps:

(1) transforming a wild type promoter region shown as SEQ ID NO.1 to mutate-303 bp nucleotides to obtain a mutated promoter region nucleotide sequence;

(2) connecting the nucleotide sequence of the mutant promoter region with a plasmid to construct a recombinant vector;

(3) and introducing the recombinant vector into a host strain to obtain the L-lysine-producing recombinant strain containing the mutated promoter region.

According to the present invention, in the step (1), the method for mutating comprises mutagenesis, PCR site-directed mutagenesis or homologous recombination, preferably PCR site-directed mutagenesis.

According to the invention, said step (1) comprises:

according to the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers for amplifying the promoter region fragment of the sdaA gene are synthesized, and the nucleotide sequence of the mutant promoter region is obtained by PCR site-specific mutagenesis

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

P1:5'CCGGAATTC ATTCGCGGATCTCCGTTTA AG 3'(EcoR I)(SEQ ID NO:3)

P2:5'CTTTACAGGG GCTGGGTCAT GTCTTG 3'(SEQ ID NO:4)

P3:5'CAAGAC ATGACCCAGC CCCTGTAAAG 3'(SEQ ID NO:5)

P4:5'ACATGCATGC AACGTCAGGG AATACGACAC 3'(Sph I)(SEQ ID NO:6)；

in one embodiment of the present invention, the step (1) comprises: carrying out PCR amplification by using Corynebacterium glutamicum ATCC13032 as a template and primers P1/P2 and P3/P4 respectively to obtain two DNA fragments with sizes of 660bp and 626bp and separated from an sdaA gene promoter region; the nucleotide sequence of the mutant promoter region (SEQ ID NO:2) was obtained by Overlap PCR amplification (Overlap PCR) using the above two DNA fragments as templates and P1 and P4 as primers.

And (2) the two ends of the DNA fragment containing the promoter region of the sdaA gene and separated from the promoter region of the sdaA gene respectively contain EcoR I and Sph I enzyme cutting sites. The DNA fragment will result in a mutation of the nucleotide at position-303 bp of the promoter region of the sdaA gene from adenine (A) to cytosine (C) in the host strain (e.g., YP 097158).

According to the invention, said step (2) comprises: and (3) carrying out agarose gel electrophoresis and separation and purification on the product amplified by the overlapping PCR reaction, and connecting the fragment subjected to double enzyme digestion (EcoR I/Sph I) with the shuttle plasmid subjected to the same double enzyme digestion (EcoR I/Sph I) to obtain the allelic replacement recombinant vector.

According to the invention, the shuttle plasmid is a pk18mobsacB plasmid; the constructed recombinant vector is pk18-PsdaA^A(-303)C。

In one embodiment of the invention, the recombinant plasmid has a kanamycin resistance marker.

In one embodiment of the present invention, the conversion of step (3) is an electrical conversion process; illustratively, in the step (3), the recombinant plasmid is transformed into the strain YP 097158.

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

According to the application and the method, the recombinant strain is adopted for fermentation, and the L-lysine is prepared. According to the application and method of the present invention, the recombinant strain of the present invention can be used alone, or can be used in combination with other L-lysine producing bacteria.

Advantageous effects

The recombinant strain is obtained by introducing point mutation into the wild promoter region of the sdaA gene, compared with the strain without mutation, the obtained strain further improves the yield of L-lysine, and has no conflict with the modified sites of the existing modified L-lysine-producing strains with high yield of L-lysine, so that a novel mode for improving the fermentation amount of L-lysine is realized, and the application is facilitated.

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.