CN115572704A - Recombinant microorganism and construction method and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to a recombinant microorganism and a construction method and application thereof. The present invention provides a recombinant microorganism having reduced expression and/or enzymatic activity of the mechanosensitive protein Ynai or a homologue or functional variant thereof as compared to its starting strain. The invention obviously improves the yield and the conversion rate of amino acids such as threonine by reducing the expression of the mechanosensitive protein Ynai in the microorganism. The threonine yield and the conversion rate of the recombinant microorganism with reduced expression of the mechanosensitive protein Ynai are obviously improved compared with those of the original strain, the fermentation production cost of threonine is reduced, and an effective modification target point and strain are provided for breeding of a threonine high-yield strain.

Description

Recombinant microorganism and construction method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a recombinant microorganism and a construction method and application thereof.

Background

Amino acids are the basic building blocks of proteins, many of which exert important physiological functions in the human and animal body, and many of which have become important fine chemicals in the chemical industry. Taking L-threonine as an example, it is one of 8 amino acids essential for human and animal growth, and is widely used in feed, food additives, preparation of pharmaceutical auxiliary materials, and the like.

The production of amino acids by microbial fermentation has become the main process for the production of various amino acids, and at present, L-threonine is mainly produced by microbial fermentation, and various bacteria are available for L-threonine production, for example: mutant strains obtained by mutagenesis of wild-type strains of Escherichia coli, corynebacterium, serratia, etc., including amino acid analog-resistant mutant strains or various auxotrophs such as methionine, lysine, isoleucine, etc., are used as production strains (Japanese patent application laid-open No. 224684/83; korean patent application laid-open No. 8022/87). 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.

With the increasing demand of threonine in the world, the construction and modification of high-yield threonine strains are particularly important. In the Chinese patent CN03811059.8 applied by Korean CJ corporation, 2003, the expression of thrABC, a key gene for threonine synthesis, was enhanced by deleting 39bp sequences from positions-56 to-18 of the threonine operon sequence using E.coli, and the threonine productivity was improved by 22%. Kwang Ho Lee et al (Kwang Ho Lee et al, systems metabolism engineering for L-threonine production, mol Syst biol.2007; 3. In the Chinese patent 201611250306.8 applied by the plum blossom group in 2016, MHZ-0215-2 strain is obtained by strengthening pntAB gene and heterogeneously introducing pyc gene, the threonine yield of the strain is 12.4g/L, the conversion rate is about 16.2%, and the strain has no plasmid load. The fermentation production performance of the production strain is a key factor for determining the yield, the conversion rate and the like of the amino acid, so that the development of the strain with higher fermentation production performance has important significance for improving the yield and the conversion rate of the amino acid.

Disclosure of Invention

The invention aims to provide a recombinant microorganism and a construction method and application thereof. Another object of the present invention is to provide a method for producing an amino acid or a derivative thereof using the recombinant microorganism.

Specifically, the invention provides the following technical scheme:

the present invention provides a recombinant microorganism having reduced expression and/or enzymatic activity of mechanosensitive protein Ynai or a homologue or functional variant thereof, compared to its starting strain.

Ynai is a mechanically sensitive protein and is also Na ⁺ /K ⁺ Selective proteins, belonging to the Mscs family, are mechanosensitive proteins that can sense an increase in membrane tension when cells pass from a high osmotic pressure environment to a low osmotic pressure environment, thereby releasing osmotically active solutes and ions to protect the cells from hypotonic shock.

The starting strain described above is the strain used as a starting strain before reducing the expression and/or enzymatic activity of mechanosensitive protein Ynai or a homologue or functional variant thereof. The recombinant microorganism can be obtained by subjecting the starting strain to genetic engineering or mutagenesis to reduce the expression and/or enzymatic activity of its mechanosensitive protein Ynai or a homologue or functional variant thereof.

In the invention, the mechanosensitive protein Ynai has any one of the following amino acid sequences:

(1) An amino acid sequence as shown in SEQ ID NO. 1;

(2) The amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;

(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown as SEQ ID No. 1.

In Escherichia coli (Escherichia coli), the amino acid sequence of mechanosensitive protein Ynai I is shown in SEQ ID NO.1, the ID number of the coding gene ynaI is 945898, and the nucleotide sequence is shown in SEQ ID NO. 2.

In the present invention, the reduction of the expression and/or the enzyme activity is achieved by a combination of one or more of the following (1) and (2):

(1) Inserting, deleting or replacing one or more bases of the coding gene of the mechanosensitive protein Ynai to reduce the expression amount, the enzymatic activity or inactivate the mechanosensitive protein Ynai;

(2) Replacing the transcription or translation regulatory element of the gene encoding the mechanosensitive protein Ynai with a less active regulatory element such that the expression amount thereof is reduced, the enzymatic activity is reduced or inactivated.

As a preferred embodiment of the present invention, the reduction of the expression and/or enzymatic activity of the mechanosensitive protein YnaI is achieved by inactivating said protein.

Preferably, the starting strain described above is a bacterium capable of accumulating an amino acid or a derivative thereof. The bacteria capable of accumulating amino acids or derivatives thereof may be wild-type strains or strains obtained by genetic engineering, mutagenesis. The starting strain of the present invention is not particularly limited with respect to the yield of the amino acid or the derivative thereof.

In particular, the starting strain preferably contains one or more of the following mutations:

(1) Enhanced expression of the pntAB gene;

(2) Expressing a pyc gene from Corynebacterium glutamicum;

(3) A mutant thrA expressing thrA (S345P);

(4) Knocking out tdh gene;

(5) Increase the copy number of thrA (S345P) BC.

As a preferred embodiment of the invention, the starting strain is MHZ-0215-2, which is disclosed in Chinese patent 201611250306.8 and has the following biological preservation information: and (3) classification and naming: escherichia coli (Escherichia coli) was deposited in China general microbiological culture Collection center (CGMCC) at 2016, 11, 30 days, with the accession number of CGMCC No. 3, ministry of microbiology, china academy of sciences, no. CGMCC No.13403, in North Cheng Xilu 1, the republic of Beijing.

The reduction of the expression and/or enzymatic activity of mechanosensitive protein Ynai can significantly increase the production of threonine, glycine, or isoleucine. In this regard, the amino acid according to the invention is preferably threonine, glycine or isoleucine.

The recombinant microorganism of the invention is a bacterium selected from the genera Escherichia, corynebacterium and Serratia.

Wherein, the Escherichia bacteria include but are not limited to Escherichia coli (Escherichia coli), the Corynebacterium bacteria include but are not limited to Corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium effectivum (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), corynebacterium ammoniagenes (Corynebacterium aminogenes).

Preferably, the recombinant microorganism is escherichia coli.

The invention also provides a construction method of the recombinant microorganism, which comprises the following steps: reducing the expression and/or the enzyme activity of the mechanosensitive protein Ynai in the original strain by a genetic engineering or mutagenesis method.

The above-mentioned genetic engineering or mutagenesis may be performed by a method commonly used in the art, and the genetic engineering method may be a conventional method of gene mutation or deletion. The mutagenesis method may be physical and/or chemical mutagenesis.

As an embodiment of the invention, the genetic engineering method is CRISPR/Cas9 technology. Specifically, plasmid containing sgRNA of targeted ynaI gene, upstream and downstream homologous arms of ynaI gene and Cas9 protein is introduced into a starting strain, and the ynaI gene is knocked out through homologous recombination.

The yield and the conversion rate of the amino acid (particularly threonine) or the derivative thereof of the recombinant microorganism provided by the invention are obviously improved.

Based on this, the present invention provides the use of said recombinant microorganism for the production of amino acids or derivatives thereof.

The invention also provides application of the recombinant microorganism or the construction method thereof in breeding of amino acid production strains.

In the above-mentioned application, preferably, the amino acid is threonine, glycine or isoleucine. More preferably threonine.

Based on the new function of the mechanosensitive protein Ynai discovered by the invention, the invention also provides any one of the following applications of the mechanosensitive protein Ynai or an inhibitor thereof, a coding gene of the mechanosensitive protein Ynai or an inhibitor thereof, and a biological material containing the coding gene or the inhibitor:

(1) Use for increasing the yield and/or conversion of an amino acid or derivative thereof from a microorganism;

(2) The application of the strain in constructing the production strain of the amino acid or the derivative thereof;

(3) Use in the fermentative production of an amino acid or a derivative thereof.

Preferably, said use is achieved by reducing the expression and/or enzymatic activity of said mechanosensitive protein Ynai.

The above-mentioned inhibitor is a protein, DNA or RNA capable of inhibiting the expression and/or enzymatic activity of mechanosensitive protein Ynai.

The above-mentioned biological materials include recombinant DNA, expression cassettes, vectors or microorganisms.

The present invention also provides a method for increasing the amino acid yield of a microorganism, comprising: reducing the expression and/or enzymatic activity of the mechanosensitive protein Ynai of said microorganism.

The present invention also provides a method for producing an amino acid or a derivative thereof by fermentation, which comprises the steps of culturing the recombinant microorganism and recovering the amino acid or the derivative thereof from the culture broth obtained.

Preferably, the amino acid is threonine, glycine or isoleucine. More preferably threonine.

For threonine, the fermentation medium used to culture the recombinant microorganism preferably comprises the following components: 50-90g/L of glucose, 5-15g/L of corn steep liquor, 5-15g/L of soybean meal hydrolysate, 1-2g/L of magnesium sulfate heptahydrate and KH ₂ PO4 1-2g/L, aspartic acid 10-20g/L, feSO ₄ 25-35mg/L，MnSO ₄ 25-35mg/L, thiamine 400-600 mug/L, pH 6.8-7.2.

For threonine, the seed medium used to culture the recombinant microorganism preferably comprises the following components: 25-35g/L glucose, 15-25g/L corn steep liquor, 4-6g/L soybean meal hydrolysate, 4-6g/L yeast extract and KH ₂ PO ₄ 2-3g/L, 0.5-1.0g/L magnesium sulfate heptahydrate, feSO ₄ 15-25mg/L，MnSO ₄ 15-25mg/L，pH 6.8-7.2。

The above-mentioned method for producing an amino acid or a derivative thereof by fermentation comprises: culturing the activated recombinant microorganism in a seed culture medium to obtain mature seed liquid, inoculating the seed liquid into a fermentation culture medium for culture, and recovering the amino acid or the derivative thereof from the obtained culture liquid.

The invention has the beneficial effects that: the invention obviously improves the yield and the conversion rate of amino acids such as threonine by reducing the expression of the mechanosensitive protein Ynai in the microorganism. The threonine yield and the conversion rate of the recombinant microorganism with reduced expression of the mechanosensitive protein Ynai are obviously improved compared with those of the original strain, the fermentation production cost of threonine is reduced, and an effective modification target point and strain are provided for breeding of a threonine high-yield strain.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The following examples, starting from MHZ-0215-2 (which is disclosed in patent application CN106635945A and belongs to the genus Escherichia (Escherichia) W3110), have been modified in relation to the metabolic pathway of L-threonine in E.coli and the genetic background of the starting strain MHZ-0215-2, to weaken the Gene coding for Ynai, in particular the knock-out Gene ynaI, gene ID:945898.

the CRISPR-Cas9 gene Editing technology (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015) reported by Jiang Y et al was mainly used for Genome Editing of Escherichia coli involved in the following examples.

In the following examples, the final concentration of Kanamycin (Kanamycin) in the medium was 50. Mu.g/mL, and the final concentration of spectinomycin (spectinomycin) in the medium was 50. Mu.g/mL.

In the following examples, all reagents used are commercially available.

The primer sequences used in the following examples are shown in Table 1.

Table 1 primer sequences used in the examples

The invention is further illustrated by the following examples.

EXAMPLE 1 preparation of ynaI knockout strain MHZ-0221-18

1. pTargetF-N20 (delta ynaI) plasmid and Donor DNA construction

(1) Using pTargetF plasmid as template (disclosed in Multigene Editing in the Escherichia coli Genome of the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015), selecting pTF-sgRNA-F/pTF-sgRNA-R primer pair, amplifying pTF linear plasmid with N20, assembling the linear plasmid at 37 ℃ by using seamless assembly ClonExpress kit, then transforming Trans1-T1 competent cells to obtain pTargetF-N20 (delta ynaI), and carrying out PCR identification and sequencing verification;

(2) Amplifying an upstream homology arm (1) by using a Uarm-F/Uarm-R primer pair by using a W3110 genome as a template;

(3) Taking the W3110 genome as a template, and selecting a Darm-F/Darm-R primer pair to amplify a downstream homology arm (2);

(4) And (3) amplifying an up-down fragment, which is also called Donor DNA, by using the (1) and (2) as templates and selecting a Uarm-F/Darm-R primer pair.

2. Competent cell preparation and electrotransformation

(1) Electrically transferring pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-2 competent cells (the transformation method and the competence preparation method refer to molecular clone III);

(2) Selecting single MHZ-0215-2 (pCas) colony, culturing in 5mL LB tube containing kanamycin and 10mM arabinose at 30 deg.C and 200r/min to OD ₆₅₀ After 0.8, electroporation competent cells were prepared (see molecular clone III).

(3) pTargetF-N20 (. DELTA.ynaI) plasmid and Donor DNA were simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5kV, 200. Omega., 25. Mu.F), spread on LB plate containing spectinomycin and kanamycin, and incubated at 30 ℃ until single colonies were visible.

3. Recombination verification

(1) Performing colony PCR amplification on the single colony by using a primer pair ynaI-F/ynaI-R;

(2) The amplification product was sent to sequencing to verify the integrity of the sequence.

4. Construction of recombinant bacteria with associated plasmid loss

(1) Selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and 0.5mM IPTG (isopropyl-beta-thiogalactoside) with final concentration, culturing overnight at 30 ℃, and streaking on an LB plate containing kanamycin;

(2) Picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, culturing overnight at 30 ℃, if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, and growing on the LB plate containing kanamycin, indicating that pTargetF-N20 (delta-ynaI) plasmid is lost;

(3) Selecting positive colonies lost by pTargetF-N20 (delta ynaI) plasmid, inoculating the positive colonies in an anti-LB-free test tube, culturing at 42 ℃ for 8 hours, streaking on an LB plate, and culturing at 37 ℃ overnight;

(4) Single colonies were picked and spotted on both kanamycin-containing LB plates and non-resistant LB plates, and if they could not grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that pCas plasmid was lost, resulting in MHZ-0221-18 strain.

The threonine producing genetically modified strains obtained in example 1 are shown in Table 2.

TABLE 2 genetically engineered bacterium constructed in example 1

Strain numbering	Genotype(s)
		MHZ-0215-2	W3110(thrA*(S345P),tdh::thrA*BC,Ptac-pntAB,IS4::P1-pyc)
MHZ-0221-18	W3110(thrA*(S345P),tdh::thrA*BC,Ptac-pntAB,IS4::P1-pyc,ΔynaI

Example 2 verification of shake flask fermentation of L-threonine producing genetically engineered bacteria

The shake flask fermentation verification of L-threonine production is carried out on the recombinant strain MHZ-0221-18 constructed in the example 1 and the starting strain MHZ-0215-2 thereof, and the details are as follows:

1. taking 2 MHZ-0215-2 and MHZ-0221-18 strains from a frozen tube, marking and activating on an LB (Langmuir-Blodgett) plate, and culturing at 37 ℃ for 24 hours;

2. the cells were scraped from the plate and inoculated into a shake flask containing 50mL of seed medium (see Table 3) and cultured at 37 ℃ and 220rpm for about 5 hours to OD ₆₅₀ Controlling the content within 1.7;

3. transferring 1mL of the seed solution into a shake flask containing 50mL of a fermentation medium (shown in Table 4), performing fermentation culture at 135rpm in a reciprocating shaking table at 37 ℃ until residual sugar is exhausted, and measuring OD (optical density) of a sample after fermentation is finished ₆₅₀ And the content of L-threonine was measured by HPLC, and the amount of residual sugar was measured by biosensing. To ensure the reliability of the experiment, the flask was subjected to 3 replicates and the average threonine production and sugar acid conversion results are shown in table 5.

TABLE 3 seed culture Medium

Composition (I)	Concentration of
		Glucose	30g/L
Corn steep liquor	20g/L
		Soybean meal hydrolysate	5g/L
Yeast cream	5g/L
		KH 2 PO 4	2.5g/L
Magnesium sulfate heptahydrate	0.7g/L
		FeSO 4 、MnSO 4	20mg/L
pH	7.0

TABLE 4 fermentation Medium

Composition (A)	Concentration of
		Glucose	70g/L
Corn steep liquor	10g/L
		Soybean meal hydrolysate	10g/L
Magnesium sulfate heptahydrate	1.5g/L
		KH 2 PO 4	1.5g/L
Aspartic acid	15g/L
		FeSO 4	30mg/L
MnSO 4	30mg/L
		Thiamine	500μg
pH	7.0

TABLE 5 comparison of the Productivity of threonine-producing genetically engineered bacteria

As can be seen from Table 5, the L-threonine yield of the novel Escherichia coli MHZ-0221-18 is higher than that of the control strain MHZ-0215-2, and the average conversion rate of shake flask fermentation of the modified strain MHZ-0221-18 is 18.26%, which is 2.05% higher than that of the original strain. The shake flask fermentation result shows that the threonine production capacity of the modified bacteria MHZ-0221-18 is obviously superior to that of the developed bacteria MHZ-0215-2. Therefore, the knockout of the gene ynaI can obviously improve the threonine production capacity of the strain.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims (10)

1. A recombinant microorganism having a reduced expression and/or enzymatic activity of the mechanosensitive protein YnaI or a homologue or functional variant thereof, as compared to its starting strain.

2. The recombinant microorganism according to claim 1, wherein the mechanosensitive protein Ynai has any one of the following amino acid sequences:

(1) An amino acid sequence shown as SEQ ID NO. 1;

(2) The amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;

(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown as SEQ ID No. 1.

3. The recombinant microorganism according to claim 1 or 2, wherein the reduction in expression and/or enzyme activity is achieved by a combination of one or more of the following (1), (2):

(1) Inserting, deleting or replacing one or more bases of the coding gene of the mechanosensitive protein Ynai to reduce the expression amount, the enzymatic activity or inactivate the mechanosensitive protein Ynai;

(2) Replacing a transcription or translation regulatory element of the gene encoding the mechanosensitive protein Ynai with a less active regulatory element such that the expression amount thereof is reduced, the enzymatic activity is reduced or inactivated;

preferably, the reduction of the expression and/or enzymatic activity of the mechanosensitive protein Ynai is achieved by inactivation of said protein.

4. The recombinant microorganism according to any one of claims 1 to 3, wherein the starting strain is a bacterium capable of accumulating an amino acid or a derivative thereof;

preferably, the amino acid is threonine, glycine or isoleucine.

5. The recombinant microorganism according to any one of claims 1 to 4, wherein the recombinant microorganism is a bacterium selected from the group consisting of the genus Escherichia, corynebacterium, and Serratia;

preferably, the recombinant microorganism is escherichia coli.

6. The method for constructing a recombinant microorganism according to any one of claims 1 to 5, comprising: reducing the expression and/or the enzyme activity of the mechanosensitive protein Ynai in the original strain by a genetic engineering or mutagenesis method.

7. Use of a recombinant microorganism according to any one of claims 1 to 5 for the production of an amino acid or a derivative thereof;

preferably, the amino acid is threonine, glycine or isoleucine.

8. Use of the recombinant microorganism according to any one of claims 1 to 5 or the method of constructing according to claim 6 for breeding an amino acid-producing strain;

preferably, the amino acid is threonine, glycine or isoleucine.

9. Any one of the following applications of mechanosensitive protein Ynai or an inhibitor thereof, a coding gene of mechanosensitive protein Ynai or an inhibitor thereof, and a biological material containing the coding gene or the inhibitor:

(1) Use for increasing the yield and/or conversion of an amino acid or derivative thereof from a microorganism;

(2) The application of the strain in constructing the production strain of the amino acid or the derivative thereof;

(3) Use in the fermentative production of an amino acid or a derivative thereof;

preferably, said use is achieved by reducing the expression and/or enzymatic activity of said mechanosensitive protein Ynai.

10. A method for producing an amino acid or a derivative thereof by fermentation, comprising culturing the recombinant microorganism according to any one of claims 1 to 5, and recovering the amino acid or the derivative thereof from the obtained culture solution.