CN115612680A

CN115612680A - Recombinant microorganism for producing threonine, method for constructing the same, and method for producing threonine using the same

Info

Publication number: CN115612680A
Application number: CN202110784728.8A
Authority: CN
Inventors: 刘涛; 康培; 王治权; 赵津津; 李岩
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-07-12
Filing date: 2021-07-12
Publication date: 2023-01-17

Abstract

The invention relates to the technical field of microorganisms, in particular to a recombinant microorganism for producing threonine, a construction method thereof and a method for producing threonine by using the recombinant microorganism. The invention finds that the expression of beta-phosphoglucomutase YcjU in the microorganism is reduced, and the yield and the conversion rate of amino acids such as threonine can be obviously improved. The recombinant microorganism with the reduced beta-phosphoglucomutase YcjU expression has the advantages that the threonine yield and the conversion rate 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 the breeding of threonine high-yield strains.

Description

Recombinant microorganism for producing threonine, method for constructing the same, and method for producing threonine using the same

Technical Field

The invention relates to the technical field of microorganisms, in particular to a recombinant microorganism for producing threonine, a construction method thereof and a method for producing threonine by using the recombinant microorganism.

Background

L-threonine is an intermediate metabolite, can be used as an important precursor for the synthesis of many amino acids, and is an essential amino acid indispensable to the human body. Threonine is widely used in the industries of food, feed, medicine and the like. The production method of threonine mainly comprises a fermentation method, a protein hydrolysis method and a chemical synthesis method, and the microbial fermentation method is the mainstream production method of threonine at present. For the production of L-threonine, mutant strains derived from escherichia coli wild-type strains, corynebacterium sp, serratia sp, or Providencia sp are used. In the method for producing L-threonine using a mutant strain, japanese patent No. 10037/81 discloses a method for using a microorganism belonging to the species Escherichia coli, having diaminopimelic acid and methionine auxotrophic phenotypes, and having L-threonine biosynthesis not affected by feedback inhibition by threonine by mutation.

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. The screening of the high-producing strains by the gene editing method is more advantageous, for example: chinese patent CN 111019878A knocks out the key gene dapA of the competitive pathway of L-threonine synthesis and the threonine dehydrogenase coding gene tdh through the CRISPR-Cas9 gene editing technology, so that the yield of threonine reaches 120g/L.

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 CJ corporation of Korea 2003, the expression of thrABC, a key gene for threonine synthesis, was enhanced by deletion of 39bp sequence from-56 to-18 of threonine operon sequence using Escherichia coli, and threonine productivity was improved by 22%. In the Chinese patent CN1654634A filed by CJ corporation in Korea in 2005, the production of threonine was increased by 18% -25% by inactivating GalR gene.

The fermentation production performance of the amino acid production strain is a key factor for determining the yield, the conversion rate and the like of the amino acid, so that the improvement of the fermentation production performance of the production strain has important significance for the improvement of the yield and the conversion rate of the amino acid and the further reduction of the cost.

Disclosure of Invention

The present invention aims to provide a recombinant microorganism producing amino acids such as threonine, a method for constructing the same, and a method for producing amino acids such as threonine.

Specifically, the invention provides the following technical scheme:

the present invention provides any one of the following applications of beta-phosphoglucomutase YcjU or an inhibitor thereof, beta-phosphoglucomutase gene YcjU or an inhibitor thereof, and a biomaterial containing said gene YcjU or said inhibitor:

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

(2) The application in constructing amino acid producing strain;

(3) The application in the fermentation production of amino acid.

In particular, the use is achieved by reducing the expression and/or enzymatic activity of the beta-phosphoglucomutase YcjU.

The present invention finds that the yield and conversion rate of amino acids can be significantly improved by reducing the expression and/or enzymatic activity of beta-phosphoglucomutase YcjU in microorganisms.

YcjU is a gene encoding beta-phosphoglucomutase, and The interconversion of glucose-1-phosphate and glucose-6-phosphate is a key step in The metabolism of all cells, and is performed by phosphoglucomutase (E.C.2.7.5.1) (Ray WJ Jr, peck EJ Jr (1972) phosphoglucutases. In: boyer PD (ed) The Enzymes, vol.6.Academic Press, new York, pp 407-458). When the carbon source is galactose, glucose-1-phosphate produced by galactose metabolism is converted to glucose-6-phosphate by phosphoglucomutase. When the carbon source is other than galactose, the primary function of phosphoglucomutase is to convert glucose-6-phosphate to glucose-1-phosphate. Glucose-6-phosphate is a common intermediate product of glycolysis, aerobic oxidation, pentose phosphate pathway, and glycogen synthesis and decomposition pathways, and is a cross-point of each metabolic pathway.

In the above applications, the inhibitor is a protein, DNA or RNA capable of inhibiting the expression and/or enzymatic activity of β -phosphoglucomutase YcjU.

In the above applications, the biological material includes recombinant DNA, an expression cassette, a vector or a microorganism.

In the present invention, the expression and/or reduction of the enzymatic activity of β -phosphoglucomutase YcjU can be achieved by a combination of one or more of the following (1) and (2):

(1) Inserting, deleting or replacing one or more bases of a gene encoding the beta-phosphoglucomutase YcjU so as to reduce the expression amount, the enzyme activity or inactivate the beta-phosphoglucomutase YcjU;

(2) Replacing a transcription or translation regulatory element of the gene encoding beta-phosphoglucomutase YcjU with a less active regulatory element so that the expression amount thereof is reduced, the enzymatic activity is reduced or inactivated.

Preferably, the reduction of expression and/or enzymatic activity of the beta-phosphoglucomutase YcjU is achieved by inactivation of said enzyme.

In one embodiment of the present invention, the reduction of expression and/or enzymatic activity of the beta-phosphoglucomutase gene ycjU is achieved by deletion thereof.

In the present invention, the beta-phosphoglucomutase YcjU 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 beta-phosphoglucomutase YcjU is shown as SEQ ID NO.1, and the nucleotide sequence of coding gene ycjU is shown as SEQ ID NO. 2.

The present invention also provides a recombinant microorganism having reduced expression and/or enzymatic activity of beta-phosphoglucomutase YcjU or a homologue or functional variant thereof as compared to its starting strain.

Wherein the reduction of expression and/or enzyme activity is achieved by one or more of the following (1) and (2):

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

The starting strain according to the invention is the strain used as a starting strain before reducing the expression and/or the enzymatic activity of beta-phosphoglucomutase YcjU or a homologue or a 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 beta-phosphoglucomutase YcjU or a homologue or functional variant thereof.

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.

The amino acid according to the invention is preferably an L-amino acid. The expression of beta-phosphoglucomutase YcjU and/or the reduction in enzyme activity 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 starting strain according to the invention preferably contains one or more of the following mutations:

(1) Enhanced expression of the pntAB gene;

(2) Expressing the 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.

The starting strain is selected from bacteria of 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 starting strain is escherichia coli.

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 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 beta-phosphoglucomutase YcjU 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, plasmids containing sgrnas targeting the ycjU gene, upstream and downstream homology arms of the ycjU gene, and Cas9 protein were introduced into the starting strain, and the ycjU gene was knocked out by 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 the recombinant microorganism in the production of amino acids or derivatives thereof or in the breeding of amino acid producing strains.

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

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 YcjU of said microorganism.

The present invention also provides a method for the fermentative production of an amino acid or a derivative thereof, which comprises: culturing the recombinant microorganism and recovering the amino acid or the derivative thereof from the obtained culture solution.

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: 70-100g/L glucose, 5-10g/L corn steep liquor, 5-10g/L soybean meal hydrolysate, 0.2-0.8g/L magnesium sulfate heptahydrate and KH ₂ PO4 0.5-1.5g/L, aspartic acid 5-15g/L, feSO ₄ 25-35mg/L，MnSO ₄ 25-35mg/L, 40-60 mug/L biotin, 400-600 mug/L thiamine and 6.8-7.2 pH.

For threonine, the seed medium used to culture the recombinant microorganism preferably comprises the following components: 20-30g/L glucose, 20-30g/L corn steep liquor, 6-9g/L soybean meal hydrolysate, 2-3g/L yeast extract and KH ₂ PO ₄ 1-2g/L, 0.2-0.8g/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 beta-phosphoglucomutase YcjU in the microorganism. The threonine yield and the conversion rate of the recombinant microorganism with the reduced expression of the beta-phosphoglucomutase YcjU are obviously improved compared with those of the original strain, the fermentation production cost of threonine is favorably reduced, and an effective modification target point and strain are provided for the breeding of threonine high-yield strains.

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 belonging to the genus Escherichia (Escherichia) W3110), were modified in relation to the genome of MHZ-0215-2 to attenuate the expression of the ycjU gene of the strain, mainly by knocking out the ycjU gene, and thereby reducing the conversion of glucose-6-phosphate to glucose-1-phosphate.

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 E.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 a Strain inactivating the ycjU Gene MHZ-0221-12

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

(1) Using pTargetF plasmid as template (from Multigene Editing in the Escherichia coli Genome via 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 (ycjU), and carrying out PCR identification and sequencing verification;

(2) Using a W3110 genome as a template, and selecting a ycjU-UF/ycjU-UR primer pair to amplify an upstream homology arm (1);

(3) Using a W3110 genome as a template, and selecting a ycjU-DF/ycjU-DR primer pair to amplify a downstream homology arm (2);

(4) And (3) selecting ycjU-UF/ycjU-DR primer pair by taking the (1) and (2) as templates to amplify an up-down full-length fragment, which is also called Donor DNA.

2. Competent cell preparation and electrotransformation

(1) (ii) electrotransfering pCas plasmid (derived from Multigene differentiation 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 competent preparation method refer to molecular clone III);

(2) A single MHZ-0215-2 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD ₆₅₀ After 0.4, electroporation competent cells were prepared (see "molecular clone III" for a competent preparation method).

(3) pTargetF-N20 (ycjU) plasmid and the Donor DNA constructed in (1) 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 a single colony was visible.

3. Recombination verification

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

(2) And (3) amplifying a target fragment by using a primer pair ycjU-F/ycjU-R, and sequencing an amplification product to verify the integrity of the sequence.

4. Construction of the relevant 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 (ycjU) plasmid is lost;

(3) Selecting positive colonies lost by pTargetF-N20 (ycjU) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight;

(4) Single colonies were picked as spots on both kanamycin-containing LB plates and on non-resistant LB plates, and if they failed to grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that the pCas plasmid was lost, giving rise to ycjU knock-out strain MHZ-0221-12 (Table 2).

TABLE 2 genetically engineered bacteria constructed in this example

Strain numbering	Genotype(s)
		MHZ-0221-12	MHZ-0215-2-ΔycjU

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

The shake flask fermentation verification of L-threonine production was carried out on the strain MHZ-0221-12 constructed in example 1 and the starting strain MHZ-0215-2, and the details are as follows:

1. taking 2 strains of MHZ-0215-2 and MHZ-0221-12 from a frozen tube, streaking and activating on an LB (Langmuir-Blodgett) plate, and culturing at 37 ℃ for 18-24h;

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 90rpm for about 5 hours to OD ₆₅₀ Controlling the content within 2;

3. transferring 2mL of the seed solution into a shake flask containing 20mL of a fermentation medium (shown in Table 4), performing fermentation culture at 100rpm with a reciprocating shaking table at 37 ℃ until residual sugar is exhausted, and measuring OD 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, 3 replicates of the shake flask fermentation were run and the average acid yield and conversion results are shown in table 5.

TABLE 3 seed culture Medium

TABLE 4 fermentation Medium

Composition (I)	Concentration of
		Glucose	85g/L
Corn steep liquor	6g/L
		Soybean meal hydrolysate	7.7g/L
Magnesium sulfate heptahydrate	0.5g/L
		KH ₂ PO ₄	1.0g/L
Aspartic acid	10g/L
		FeSO ₄ 、MnSO ₄	30mg/L
Biotin	50μg
		Thiamine	500μg
pH	7.2

TABLE 5 comparison of productivity of threonine-producing genetically engineered bacteria

As can be seen from Table 5, the L-threonine yield of the MHZ-0221-12 strain is significantly higher than that of the original strain MHZ-0215-2, the threonine yield of the MHZ-0215-12 is 13.83g/L, the sugar-acid conversion rate is 16.4%, the yield is 14.3% higher than that of the original strain, and the conversion rate is 15.5%. The shake flask fermentation experiment result shows that the knockout of the gene of the coliform strain ycjU can obviously improve the threonine production capacity of the strain.

Example 3 fermentation tank fermentation validation of L-threonine-producing genetically engineered bacteria

The fermentation verification of the 1L fermentation tank for producing L-threonine is carried out on the strain MHZ-0221-12 constructed in the example 1 and the starting strain MHZ-0215-2, and the details are as follows:

1. 2 strains of MHZ-0215-2 and MHZ-0221-12 are taken from a freezing storage tube, streaked and activated on an LB plate, and cultured for 18-24h at 37 ℃;

2. the cells were scraped from the plate and inoculated into a shake flask containing 100mL of seed medium (see Table 3) and cultured at 37 ℃ and 90rpm for about 5 hours at OD ₆₅₀ Controlling the content within 2;

3. 1L tank fermentation preparation work: pH correction, pump correction, in-situ cleaning and dissolved oxygen correction.

4. Transferring 60mL of seed solution into 1L fermentation tank containing 360mL of fermentation medium (see Table 4), performing fermentation culture at 37 deg.C at 500-1200 rpm until residual sugar is exhausted, and measuring OD of sample after fermentation 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 fermentation tank was subjected to 3 replicates and the average results of acid production and conversion are shown in table 6.

TABLE 6 comparison of productivity of threonine-producing genetically engineered bacteria

As can be seen from Table 6, the L-threonine yield of the MHZ-0221-12 strain is significantly higher than that of the original strain, the L-threonine yield of the MHZ-0215-12 strain is 19g/L, the saccharic acid conversion rate is 22.3%, the yield is increased by 42.8% compared with that of the original strain, and the conversion rate is increased by 42.9%. Fermentation tank fermentation experiment results show that the knockout of the gene of the coliform strain ycjU can remarkably improve the threonine production capacity of the strain.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

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Claims

1. Any one of the following applications of beta-phosphoglucomutase YcjU or an inhibitor thereof, beta-phosphoglucomutase gene ycjU or an inhibitor thereof, and a biological material containing the gene ycjU or the inhibitor:

(2) The application in constructing amino acid producing strain;

(3) The application in the fermentation production of amino acid.

2. The use according to claim 1, wherein the use is by decreasing the expression and/or enzymatic activity of the beta-phosphoglucomutase YcjU.

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

4. The use according to any one of claims 1 to 3, wherein the beta-phosphoglucomutase YcjU has any one of the following amino acid sequences:

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

5. A recombinant microorganism having reduced expression and/or enzymatic activity of β -phosphoglucomutase YcjU or a homologue or functional variant thereof as compared to the starting strain.

6. The recombinant microorganism according to claim 5, wherein the reduction of expression and/or enzyme activity is achieved by a combination of one or more of the following (1), (2):

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

7. The recombinant microorganism according to claim 5 or 6, wherein the starting strain is a bacterium capable of accumulating an amino acid or a derivative thereof, and the bacterium is selected from the group consisting of the genera Escherichia, corynebacterium, and Serratia;

preferably, the amino acid is threonine, glycine or isoleucine, and the starting strain is Escherichia coli.

8. The method for constructing a recombinant microorganism according to any one of claims 5 to 7, which comprises: reducing the expression and/or the enzyme activity of the beta-phosphoglucomutase YcjU in the starting strain by a genetic engineering or mutagenesis method.

9. Use of the recombinant microorganism according to any one of claims 5 to 7 for the production of an amino acid or a derivative thereof or for the selective breeding of an amino acid-producing strain;

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

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 5 to 7, and recovering the amino acid or the derivative thereof from the obtained culture solution.