Recombinant microorganism and use thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a recombinant microorganism and application thereof. The recombinant microorganism comprises a trmH gene mutant, wherein the mutant is obtained by mutating alanine at the 84th amino acid of the amino acid sequence of the trmH gene into valine. The present application finds that the mutant of the trmH gene of a strain has a significantly improved ability to produce amino acids, especially the level of producing L-threonine. In addition, the recombinant microorganism provided by the present application also has high temperature stability, and still has a high acid production level under the condition of large temperature fluctuation, which is of great significance in the field of promoting the production of threonine by microorganisms.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a recombinant microorganism and its applications. Background Technology

[0002] L-Threonine is one of the eight essential amino acids for human and animal growth, and it is widely used in feed, food additives, and pharmaceutical excipients. Currently, L-Threonine is mainly produced through microbial fermentation. Various bacteria can be used for L-Threonine production, such as wild-type mutant strains induced from *Escherichia coli*, *Corynebacterium*, and *Serratia* as production strains. Specific examples include amino acid analogue-resistant mutants or auxotrophic strains such as those containing methionine, lysine, and isoleucine (Japanese Patent Application Publication No. 224684 / 83; Korean Patent Application Publication No. 8022 / 87). However, traditional mutagenesis breeding, due to random mutations, results in slow strain growth and the production of numerous byproducts, making it difficult to obtain high-yielding strains.

[0003] With the increasing global demand for threonine, the construction and modification of high-yield threonine-producing strains are particularly important. In Chinese patent CN03811059.8, filed in 2003 by CJ Corporation of South Korea, a method was proposed using *E. coli* to enhance the expression of the key threonine synthesis gene *thrABC* by deleting a 39bp sequence from position -56 to -18 of the threonine operon sequence, resulting in a 22% increase in threonine productivity. Kwang Ho Lee et al. (Kwang Ho Lee et al., Systems metabolic engineering of Escherichia coli for L-threonine production, Mol Syst Biol. 2007; 3:149) used systems metabolic engineering strategies to remove product feedback inhibition by mutating the genes thrA and lysC encoding aspartate kinases I and III, knocking out tdh and weakening ilvA to remove byproducts glycine and isoleucine, and providing more precursors for threonine synthesis by inactivating the competitive pathway genes metA and lysA. The resulting TH28C (pBRThrABCR3) strain produced 82.4 g / L of acid after 50 h of fermentation, with a sugar-acid conversion rate of 39.3%. In 2020, Meihua Group applied for Chinese patent 201611250306.8, which obtained strain MHZ-0215-2 by strengthening the pntAB gene and heterologously introducing the pyc gene. This strain has a threonine yield of 12.4 g / L, a conversion rate of about 16.2%, and no plasmid burden.

[0004] Threonine can be degraded intracellularly to produce glycine and isoleucine. Therefore, those skilled in the art can enhance isoleucine synthesis by strengthening genes in the threonine-to-isoleucine biosynthetic pathway. Thus, methods that improve threonine yield are beneficial to the production of its downstream product, isoleucine. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides a recombinant microorganism and its application.

[0006] In a first aspect, the present invention provides an application of a trmH gene mutant in regulating the ability of a strain to produce amino acids; the trmH gene mutant is obtained by mutating alanine at position 84 of the amino acid sequence of the trmH gene to valine.

[0007] Furthermore, the amino acid sequence of the trmH gene includes the sequence shown in SEQ ID NO.3.

[0008] Furthermore, the trmH gene mutant is encoded by the nucleotide sequence shown in SEQ ID NO.5, or a nucleotide sequence having at least 60% sequence identity with SEQ ID NO.5.

[0009] Accordingly, the amino acid sequence of the trmH gene mutant is shown in SEQ ID NO.6.

[0010] Furthermore, the strain is Escherichia coli, Corynebacterium glutamicum, or Serratia marcescens; the amino acid is threonine; and the ability of the strain to produce amino acids is to enhance the strain's ability to produce threonine.

[0011] Secondly, the present invention provides a recombinant microorganism, the recombinant microorganism including the trmH gene mutant mentioned in the application.

[0012] Furthermore, the expression of the dbpA gene in the recombinant microorganism is suppressed; preferably, the dbpA gene contains the nucleotide sequence shown in SEQ ID NO.2 (the amino acid sequence encoded by it is shown in SEQ ID NO.4).

[0013] Furthermore, the starting strain of the recombinant microorganism is Escherichia coli, Corynebacterium glutamicum, or Serratia marcescens.

[0014] Furthermore, the recombinant microorganism is *Escherichia coli*, preferably, the starting strain of the recombinant microorganism is MHZ-0215-2, with the accession number CGMCC No. 13403. This strain is commercially available.

[0015] This invention obtained a high-threonine-producing mutant strain by UV mutagenesis of W3110, and whole-genome sequencing revealed mutations in its trmH and dbpA genes. To further verify whether this mutation is beneficial to threonine production, the mutation was introduced into the threonine-producing strain MHZ-0215-2, and it was found that the threonine yield was increased. When the two were combined, the performance was further improved. Therefore, it was determined that the mutation at this site is beneficial to the increase of threonine production. Furthermore, the strain can still maintain good performance when the temperature fluctuates greatly during fermentation, indicating that the stability of the strain is improved.

[0016] The present invention further provides a microbial preparation comprising the recombinant microorganism.

[0017] The present invention further provides the application of the recombinant microorganism or the microbial preparation in the production of amino acids, preferably in the production of threonine.

[0018] Thirdly, the present invention provides a method for improving the amino acid production capacity of a bacterial strain, comprising:

[0019] The alanine at position 84 of the trmH gene in the strain was mutated to valine, and / or the expression of the dbpA gene in the strain was inhibited.

[0020] Furthermore, the dbpA gene contains a nucleotide sequence as shown in SEQ ID NO.2.

[0021] Furthermore, the expression level of the dbpA gene in the strain was reduced as follows:

[0022] This inactivates the dbpA gene in the strain.

[0023] Furthermore, the strain described is *Escherichia coli*, *Corynebacterium glutamicum*, or *Serratia marcescens*. This invention has the following beneficial effects:

[0024] This invention provides a trmH gene mutant, in which the ability to produce amino acids is significantly enhanced when the trmH gene in a strain undergoes a corresponding mutation. Furthermore, by simultaneously knocking out the dbpA gene in the microorganism, a recombinant microorganism is obtained. This recombinant microorganism exhibits significantly enhanced L-threonine production, higher temperature stability, and greater adaptability to temperature fluctuations, maintaining high acid production capacity even under significant temperature variations.

[0025] This demonstrates that the acid production capacity and temperature adaptability of the strain can be improved by modifying two genes, trmH and dpbA, which is of great significance for enhancing the strain's ability to produce threonine. Detailed Implementation

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0027] This invention uses MHZ-0215-2 (strain accession number CGMCC No. 13403, disclosed in Chinese Patent 201611250306.8) as the starting strain and performs relevant modifications on its genome. The main methods are trmH point mutation and / or dbpA deletion.

[0028] The genome editing of Escherichia coli mainly draws on the CRISPR-Cas9 gene editing technology reported by Jiang Y et al. (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015).

[0029] In the following examples, the final concentration of kanamycin in the culture medium was 50 μg / mL, and the final concentration of spectinomycin in the culture medium was 50 μg / mL.

[0030] In the following examples, all reagents used were commercially available. The parent strain of the high-conversion threonine production strain provided by this invention is MHZ-0215-2, belonging to W3110 (Escherichia).

[0031] The primer sequences used in the examples are shown in the table below:

[0032] Table 1 shows the primer sequences used in the examples.

[0033]

[0034]

[0035] Example 1: Preparation of strain MHZ-0221-15 with cytosine replaced by thymine at nucleotide position 251 of trmH

[0036] 1. Construction of pTargetF-N20(trmH C251T) plasmid and Donor DNA

[0037] Step 1: Using pTargetF plasmid as a template (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), the pTF-sgRNA-trmH-F / pTF-sgRNA-trmH-R primer pair was selected to amplify the pTF linear plasmid containing N20. This linear plasmid was assembled at 37℃ using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain pTargetF-N20(trmH C251T), which was then identified by PCR and sequenced for verification.

[0038] Step 2: Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the trmH-UF / trmH-UR primer pair;

[0039] Step 3: Using the W3110 genome as a template, downstream homologous arms ② were amplified using the trmH-DF / trmH-DR primer pair;

[0040] Step 4: Using ① and ② as templates, select the trmH-UF / trmH-DR primer pair to amplify the full-length trmH C251T fragment, also known as Donor DNA (the nucleotide sequence of trmH itself is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.3; the nucleotide sequence of trmH C251T is shown in SEQ ID NO.5, and the encoded protein is shown in SEQ ID NO.6).

[0041] 2. Preparation and electroporation of competent cells

[0042] Step 1: Electroporate the pCas plasmid (from the literature 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 competent cell preparation method are both based on Molecular Cloning III);

[0043] Step 2: Pick a single colony of MHZ-0215-2 (pCas) and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 rpm until OD500.650 Electrocompetent cells were prepared after 0.4% concentration (the method for preparing competent cells is described in Molecular Cloning III).

[0044] Step 3: Electroporate the pTargetF-N20(trmH C251T) plasmid and the Donor DNA constructed in Step 1 into MHZ-0215-2(pCas) competent cells simultaneously (electroporation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30℃ until single colonies are visible.

[0045] 3. Recombination Verification

[0046] Step 1: Use primer pair trmH-F / trmH-R to perform colony PCR verification on the above single colonies;

[0047] Step 2: Amplify the target fragment using primer pair trmH-F / trmH-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0048] 4. Loss of constructing related plasmids

[0049] Step 1: Select a single colony that has been correctly sequenced and inoculate it into a 5 mL LB tube containing kanamycin and a final concentration of 0.5 mM IPTG. After incubating overnight at 30°C, streak the colony onto an LB agar plate containing kanamycin.

[0050] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone, and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin and spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20(trmH C251T) plasmid has been lost.

[0051] Step 3: Pick positive colonies that have lost the pTargetF-N20(trmH C251T) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃.

[0052] Step 4: Select a single colony and spot it onto LB agar plates containing kanamycin and LB agar plates without antibiotics. If it cannot grow on LB agar plates containing kanamycin but grows on LB agar plates without antibiotics, it indicates that the pCas plasmid is lost, and the strain MHZ-0221-6 (trmHC521T) is obtained.

[0053] Example 2: Preparation of dbpA-inactivated strain MHZ-0221-16

[0054] 1. Construction of pTargetF-N20 (dpbA inactivated) plasmid and Donor DNA

[0055] Step 1: Using pTargetF plasmid as a template (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), the pTF-sgRNA-dbpA-F / pTF-sgRNA-dbpA-R primer pair was selected to amplify the pTF linear plasmid containing N20. This linear plasmid was assembled at 37℃ using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain pTargetF-N20(dbpA), which was then identified by PCR and sequenced for verification.

[0056] Step 2: Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the dbpA-UF / dbpA-UR primer pair;

[0057] Step 3: Using the W3110 genome as a template, downstream homologous arms ② were amplified using the dbpA-DF / dbpA-DR primer pair;

[0058] Step 4: Using ① and ② as templates, select the dbpA-UF / dbpA-DR primer pair to amplify the full-length fragment inactivated by dbpA, also known as Donor DNA.

[0059] 2. Preparation and electroporation of competent cells

[0060] Step 1: Electroporate the pCas plasmid (from the literature 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 competent cell preparation method are both based on Molecular Cloning III);

[0061] Step 2: Pick a single colony of MHZ-0215-2 (pCas) and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 rpm until... OD650 Electrocompetent cells were prepared after 0.4 μL of the solution (the method for preparing competent cells is described in Molecular Cloning III).

[0062] Step 3: Electroporate the pTargetF-N20(dbpA) plasmid and the Donor DNA constructed in Step 1 into MHZ-0215-2(pCas) competent cells simultaneously (electroporation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30℃ until single colonies are visible.

[0063] 3. Recombination Verification

[0064] Step 1: Use primer pair dbpA-F / dbpA-R to perform colony PCR verification on the above single colonies;

[0065] Step 2: Amplify the target fragment using primer pairs dbpA-F / dbpA-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0066] 4. Loss of constructing related plasmids

[0067] Step 1: Select a single colony that has been correctly sequenced and inoculate it into a 5 mL LB tube containing kanamycin and a final concentration of 0.5 mM IPTG. After incubating overnight at 30°C, streak the colony onto an LB agar plate containing kanamycin.

[0068] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20(dbpA) plasmid has been lost.

[0069] Step 3: Pick positive colonies that have lost the pTargetF-N20(dbpA) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃.

[0070] Step 4: Select a single colony and spot it onto LB agar plates containing kanamycin and LB agar plates without antibiotics. If it cannot grow on LB agar plates containing kanamycin but grows on LB agar plates without antibiotics, it indicates that the pCas plasmid is lost, and the MHZ-0221-16 (dbpA inactivated) strain is obtained.

[0071] Example 3: Construction of trmH and dbpA double mutant strain MHZ-0221-17

[0072] Based on MHZ-0221-16 prepared in Example 1, MHZ-0221-16 was modified according to the operation steps of Example 2, and the modified bacteria obtained was trmH and dbpA double mutant MHZ-0221-17.

[0073] The threonine-producing genetically modified strains obtained in Examples 1-3 are shown in Table 2:

[0074] Table 2 Genetically engineered bacteria constructed in this invention

[0075] strain number genotype MHZ-0221-15 MHZ-0215-2,trmH C251T MHZ-0221-16 MHZ-0215-2,△dbpA MHZ-0221-17 MHZ-0215-2,trmH C251T, △dbpA

[0076] Example 4: Verification of L-threonine-producing genetically engineered bacteria through shake-flask fermentation

[0077] Step 1: Take four strains of bacteria (MHZ-0215-2, MHZ-0221-15, MHZ-0221-16, and MHZ0221-9) from the cryopreservation tubes, streak them on LB plates for activation, and incubate at 37°C for 18-24 hours.

[0078] Step 2: Scrape a loopful of bacterial cells from the plate and inoculate it into a shake flask containing 50 mL of seed culture medium (see Table 3). Incubate at 37°C and 90 rpm for approximately 5 hours to allow the OD to reach its maximum. 650 Keep it below 2;

[0079] Step 3: Transfer 2 mL of seed culture to a shake flask containing 20 mL of fermentation medium (see Table 4), and ferment on a shaker at 37°C and 100 rpm until the residual sugar is exhausted. After fermentation, measure the OD of the sample. 650 The L-threonine content was determined by HPLC, and the residual sugar content was determined by a biosensor method. To ensure the reliability of the experiment, three experiments were conducted on the shake flasks at fermentation temperatures of 35℃, 37℃, and 39℃. The acid production and conversion rates are shown in Table 5.

[0080] Table 3 Seed culture medium (g / L)

[0081] Element concentration glucose 25 Corn syrup 25 Soybean meal hydrolysate 7.7 Yeast paste 2.5 <![CDATA[KH2PO4]]> 1.4 Magnesium sulfate heptahydrate 0.5 <![CDATA[FeSO4、MnSO4]]> 20mg / L pH 7.0

[0082] Table 4 Fermentation medium (g / L)

[0083] Element concentration glucose 85 Corn syrup 6 Soybean meal hydrolysate 7.7 Magnesium sulfate heptahydrate 0.5 <![CDATA[KH2PO4]]> 1.0 Aspartic acid 10 <![CDATA[FeSO4、MnSO4]]> 30mg / L Biotin 50μg Thiamine 500μg pH 7.2

[0084] Table 5 Comparison of productivity of threonine-producing genetically engineered bacteria

[0085]

[0086] Table 5 shows that when the temperature is below or above the optimal fermentation temperature of 37℃, the performance of the control strain MHZ-0215-2 significantly decreased, from the normal level of 14.10 g / L to about 10 g / L, a decrease of approximately 29%; the sugar-acid conversion rate decreased from 16.60% to about 12%, a decrease of approximately 27.71%. The three strains MHZ-0221-15, MHZ-0221-16, and MHZ-0221-17 showed improved threonine production and conversion rates at 37℃ compared to MHZ-0215-2, and at 35℃ and 39℃, the acid production and conversion rates did not significantly decrease compared to 37℃, remaining within ±1 g / L of their performance at 37℃. This fully demonstrates that the modification of the trmH and dpbA sites improved the strains' ability to produce threonine and further enhanced their stability.

[0087] Example 5: The stability of L-threonine-producing genetically engineered bacteria was verified in a 5L tank with large temperature fluctuations.

[0088] Step 1: Take four strains of bacteria (MHZ-0215-2, MHZ-0221-15, MHZ-0221-16, and MHZ0221-9) from the cryopreservation tubes, streak them on LB plates for activation, and incubate at 37°C for 18-24 hours.

[0089] Step 2: Scrape a loopful of bacterial cells from the plate and inoculate it into a shake flask containing 30 mL of seed culture medium (see Table 6). Incubate at 37°C and 90 rpm for approximately 5 hours to allow the OD to reach its maximum. 650 Keep it below 2;

[0090] Step 3: Transfer 30 mL of seed culture to a 5 L container containing 3 L of fermentation medium (see Table 7), maintain the temperature at 37 ± 2 °C, dissolved oxygen at 30%, and pH at 7, and measure the OD of the sample. 650 The L-threonine content was determined by HPLC, and the residual sugar content was determined by a biosensor method. The acid production and conversion results are shown in Table 8.

[0091] Table 6 Seed Culture Medium (g / L)

[0092] Element concentration glucose 25 Corn syrup 25 Soybean meal hydrolysate 7.7 Yeast paste 2.5 <![CDATA[KH2PO4]]> 1.4 Magnesium sulfate heptahydrate 0.5 <![CDATA[FeSO4、MnSO4]]> 20mg / L pH 7.0

[0093] Table 7 Fermentation medium (g / L)

[0094]

[0095]

[0096] Table 8 Comparison of productivity of threonine-producing genetically engineered bacteria

[0097] strain number Acid production (g / L) Sugar-acid conversion rate (%) MHZ-0215-2 20 16.66 MHZ-0221-15 30 25.00 MHZ-0221-16 28 23.33 MHZ-0221-17 35 29.17

[0098] As shown in Table 8, when the fermentation temperature was artificially controlled within a fluctuation range of 37±2℃, MHZ-0221-15, MHZ-0221-16, and MHZ-0221-17 still maintained superior performance, and their acid production capacity was significantly higher than that of the control MHZ-0215-5. This fully demonstrates that the modification of the trmH and dpbA sites improved the stability of the strains, enabling them to maintain good acid production capacity even under large temperature fluctuations.

[0099] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of a trmH gene mutant in improving the ability of Escherichia coli to produce threonine; wherein the trmH gene mutant is obtained by mutating alanine at position 84 of the amino acid sequence of the trmH gene to valine; the amino acid sequence of the trmH gene is shown in SEQ ID NO.

3.

2. A recombinant microorganism, characterized in that, The recombinant microorganism includes the trmH gene mutant described in the application of claim 1.

3. The recombinant microorganism according to claim 2, characterized in that, The recombinant microorganism is an Escherichia coli with the dbpA gene knocked out; the nucleotide sequence of the dbpA gene is shown in SEQ ID NO.

2.

4. A microbial preparation, characterized in that, The microbial preparation includes the recombinant microorganisms described in claim 2 or 3.

5. The use of the recombinant microorganism of claim 2 or 3 or the microbial preparation of claim 4 in the production of threonine.

6. A method for improving the ability of Escherichia coli to produce threonine, characterized in that, include: The alanine at position 84 of the amino acid sequence of the trmH gene in the *E. coli* was mutated to valine, and the dbpA gene in the strain was knocked out. The amino acid sequence of the trmH gene is shown in SEQ ID NO.3; The nucleotide sequence of the dbpA gene is shown in SEQ ID NO.2.

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