Recombinant microorganisms, methods of making and using the same

Abstract

The present application relates to the field of microbial technology, in particular to a recombinant microorganism and a construction method and application thereof. The present application provides a recombinant microorganism, wherein the recombinant microorganism has reduced expression and / or enzymatic activity of a mechanosensitive protein YnaI or a homolog or a functional variant thereof compared with a starting strain. The present application significantly improves the yield and conversion rate of threonine and other amino acids by reducing the expression of the mechanosensitive protein YnaI in the microorganism. The yield and conversion rate of threonine of the recombinant microorganism provided by the present application with reduced expression of the mechanosensitive protein YnaI are significantly improved compared with the starting strain, which is conducive to reducing the fermentation production cost of threonine and provides an effective modification target and strain for the breeding of a high-yield threonine strain.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to recombinant microorganisms, their construction methods, and applications. Background Technology

[0002] Amino acids are the basic building blocks of proteins. Many amino acids play important physiological functions in humans and animals, and many have become important fine chemicals in the chemical industry. For example, L-threonine is one of the eight essential amino acids for human and animal growth and is widely used in feed, food additives, and pharmaceutical excipients.

[0003] Microbial fermentation has become a major method for producing various amino acids. Currently, L-threonine is mainly produced through microbial fermentation, and various bacteria can be used for L-threonine production. For example, mutant strains obtained by mutagenesis of wild-type strains of Escherichia coli, Corynebacterium, and Serratia are used as production strains, including amino acid analog-resistant mutants or auxotrophic strains such as 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 many byproducts, making it difficult to obtain high-yielding strains.

[0004] 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. (Systems metabolic engineering of Escherichia coli for L-threonine production, Mol Syst Biol. 2007; 3:149) used a systems metabolic engineering strategy 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 inactivating the competitive pathway genes metA and lysA to provide more precursors for threonine synthesis. 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 2016, Meihua Group applied for Chinese patent 201611250306.8, which described the MHZ-0215-2 strain obtained by strengthening the pntAB gene and heterologously introducing the pyc gene. This strain produced threonine at a yield of 12.4 g / L and a conversion rate of approximately 16.2%, and it was free of plasmid burden. The fermentation performance of the production strain is a key factor determining amino acid yield and conversion rate; therefore, developing strains with high fermentation performance is of great significance for improving amino acid yield and conversion rate. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant microorganism, its construction method, and its application. Another purpose of this invention is to provide a method for producing amino acids or their derivatives using the recombinant microorganism.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant microorganism, which, compared with its originating strain, has reduced expression and / or enzyme activity of the mechanosensitive protein YnaI or its homologs or functional variants.

[0008] YnaI is a mechanosensitive protein and also a Na+ protein. + / K + Selective proteins, belonging to the MSCS family, are mechanosensitive proteins that can sense the increase in membrane tension when cells move from a hyperosmolar environment to a hypoosmolar environment. They then release osmotically active solutes and ions to protect the cells from the hypoosmolar shock.

[0009] The starting strains described above are strains used as a starting point before reducing the expression and / or enzyme activity of the mechanosensitive protein YnaI or its homologs or functional variants. The recombinant microorganisms can be obtained by genetically engineering or mutagenesis of the starting strains to reduce the expression and / or enzyme activity of the mechanosensitive protein YnaI or its homologs or functional variants.

[0010] In this invention, the mechanosensitive protein YnaI has any of the following amino acid sequences:

[0011] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0012] (2) An amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acids as shown in SEQ ID NO.1.

[0013] (3) An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO.1.

[0014] In Escherichia coli, the amino acid sequence of the mechanosensitive protein YnaI is shown in SEQ ID NO.1, and the ID number of its encoding gene ynaI is 945898, and its nucleotide sequence is shown in SEQ ID NO.2.

[0015] In this invention, the reduction in expression and / or enzyme activity is achieved through a combination of one or more of the following methods (1) and (2):

[0016] (1) Insert, delete or replace one or more bases in the gene encoding the mechanosensitive protein YnaI to reduce the expression level of mechanosensitive protein YnaI, reduce enzyme activity or inactivate it.

[0017] (2) Replace the transcriptional or translational regulatory element of the gene encoding the mechanosensitive protein YnaI with a less active regulatory element so as to reduce its expression level, reduce enzyme activity or inactivate it.

[0018] In a preferred embodiment of the present invention, the expression and / or enzyme activity of the mechanosensitive protein YnaI are reduced by inactivating it.

[0019] Preferably, the starting strain described above is a bacterium capable of accumulating amino acids or their derivatives. The bacterium capable of accumulating amino acids or their derivatives can be a wild-type strain or a strain obtained through genetic engineering or mutagenesis. The starting strain of this invention does not have particular limitations on the yield of the amino acids or their derivatives.

[0020] Specifically, the starting strain preferably contains one or more of the following mutations:

[0021] (1) Enhanced expression of the pntAB gene;

[0022] (2) Expression of the pyc gene derived from Corynebacterium glutamicum;

[0023] (3) The mutant thrA*(S345P) expressing thrA;

[0024] (4) Knock out the tdh gene;

[0025] (5) Increase the number of copies of thrA*(S345P)BC.

[0026] In a preferred embodiment of the present invention, the starting strain is MHZ-0215-2, which has been disclosed in Chinese Patent 201611250306.8. Its biodeposit information is as follows: Classification and nomenclature: Escherichia coli, deposited on November 30, 2016 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 13403.

[0027] The expression and / or decreased enzyme activity of the mechanosensitive protein YnaI can significantly increase the production of threonine, glycine, or isoleucine. Therefore, the amino acid used in this invention is preferably threonine, glycine, or isoleucine.

[0028] The recombinant microorganisms described in this invention are bacteria selected from the genera Escherichia, Corynebacterium, and Serratia.

[0029] The Escherichia spp. bacteria include, but are not limited to, Escherichia coli, and the Corynebacterium spp. bacteria include, but are not limited to, Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, and Corynebacterium aminogenes.

[0030] Preferably, the recombinant microorganism is Escherichia coli.

[0031] The present invention also provides a method for constructing the recombinant microorganism, the method comprising: reducing the expression and / or enzyme activity of the mechanosensitive protein YnaI in the starting strain by means of genetic engineering or mutagenesis.

[0032] The genetic engineering or mutagenesis methods described above can employ methods commonly used in the field. Specifically, the genetic engineering methods can utilize conventional gene mutation or deletion techniques. The mutagenesis methods can be physical and / or chemical mutagenesis.

[0033] In one embodiment of the present invention, the genetic engineering method is CRISPR / Cas9 technology. Specifically, a plasmid containing sgRNA targeting the ynaI gene, upstream and downstream homologous arms of the ynaI gene, and Cas9 protein is introduced into the starting strain, and the ynaI gene is knocked out through homologous recombination.

[0034] The present invention provides a significant improvement in the yield and conversion rate of amino acids (especially threonine) or their derivatives from recombinant microorganisms.

[0035] Based on this, the present invention provides the application of the recombinant microorganism in the production of amino acids or their derivatives.

[0036] The present invention also provides the application of the recombinant microorganism or its construction method in the selection of amino acid production strains.

[0037] In the applications described above, preferably, the amino acid is threonine, glycine, or isoleucine. More preferably, it is threonine.

[0038] Based on the novel function of the mechanosensitive protein YnaI discovered in this invention, this invention also provides any of the following applications of the mechanosensitive protein YnaI or its inhibitor, the encoding gene of the mechanosensitive protein YnaI or its inhibitor, and biological materials containing said encoding gene or said inhibitor:

[0039] (1) Application in increasing the yield and / or conversion rate of amino acids or their derivatives in microorganisms;

[0040] (2) Application in the construction of production strains for amino acids or their derivatives;

[0041] (3) Application in the fermentation production of amino acids or their derivatives.

[0042] Preferably, the application is achieved by reducing the expression and / or enzyme activity of the mechanosensitive protein YnaI.

[0043] The inhibitors mentioned above are proteins, DNA, or RNA that can inhibit the expression and / or enzyme activity of the mechanosensitive protein YnaI.

[0044] The biological materials mentioned above include recombinant DNA, expression cassettes, vectors, or microorganisms.

[0045] The present invention also provides a method for increasing the amino acid production of microorganisms, comprising: reducing the expression and / or enzyme activity of the mechanosensitive protein YnaI of the microorganisms.

[0046] The present invention also provides a method for producing amino acids or their derivatives by fermentation, the method comprising the steps of culturing the recombinant microorganism and recovering the amino acids or their derivatives from the obtained culture medium.

[0047] Preferably, the amino acid is threonine, glycine, or isoleucine. More preferably, it is threonine.

[0048] For threonine, the fermentation medium used to culture the recombinant microorganism preferably comprises the following components: glucose 50-90 g / L, corn steep liquor 5-15 g / L, soybean meal hydrolysate 5-15 g / L, magnesium sulfate heptahydrate 1-2 g / L, KH2PO4 1-2 g / L, aspartic acid 10-20 g / L, FeSO4 25-35 mg / L, MnSO4 25-35 mg / L, thiamine 400-600 μg / L, pH 6.8-7.2.

[0049] For threonine, the seed culture medium for culturing the recombinant microorganism preferably comprises the following components: glucose 25-35 g / L, corn steep liquor 15-25 g / L, soybean meal hydrolysate 4-6 g / L, yeast extract 4-6 g / L, KH2PO4 2-3 g / L, magnesium sulfate heptahydrate 0.5-1.0 g / L, FeSO4 15-25 mg / L, MnSO4 15-25 mg / L, pH 6.8-7.2.

[0050] The above-described method for fermenting to produce amino acids or their derivatives includes: first, culturing activated recombinant microorganisms in a seed culture medium to obtain a mature seed liquid; then, inoculating the seed liquid into a fermentation culture medium for further cultivation; and recovering the amino acids or their derivatives from the obtained culture medium.

[0051] The beneficial effects of this invention are as follows: By reducing the expression of the mechanosensitive protein YnaI in microorganisms, this invention significantly improves the yield and conversion rate of amino acids such as threonine. The recombinant microorganisms with reduced expression of the mechanosensitive protein YnaI provided by this invention have significantly higher threonine yield and conversion rate than the starting strain, which is beneficial for reducing the fermentation production cost of threonine and provides an effective modification target and strain for the breeding of high-yield threonine strains. Detailed Implementation

[0052] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0053] The following examples use MHZ-0215-2 as the starting strain (this strain has been disclosed in patent application CN106635945A, and this strain belongs to the genus Escherichia W3110). Based on the metabolic pathway of L-threonine in Escherichia coli and the genetic background of the starting strain MHZ-0215-2, relevant modifications were made to its genome to weaken the encoding gene of YnaI, specifically by knocking out the gene ynaI, Gene ID: 945898.

[0054] The genome editing of Escherichia coli involved in the following examples 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).

[0055] 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.

[0056] All reagents used in the following examples are commercially available.

[0057] The primer sequences used in the following examples are shown in Table 1.

[0058] Table 1 Primer sequences used in the examples.

[0059]

[0060] The present invention will be further illustrated below with reference to the embodiments.

[0061] Example 1: Preparation of ynaI gene knockout strain MHZ-0221-18

[0062] 1. Construction of pTargetF-N20(ΔynaI) plasmid and Donor DNA

[0063] (1) Using pTargetF plasmid as a template (published in 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-F / pTF-sgRNA-R primer pair was selected to amplify the pTF linear plasmid containing N20. The linear plasmid was assembled at 37℃ using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain pTargetF-N20(ΔynaI), which was then identified by PCR and sequenced for verification.

[0064] (2) Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the Uarm-F / Uarm-R primer pair;

[0065] (3) Using the W3110 genome as a template, the downstream homologous arm ② was amplified by using the Darm-F / Darm-R primer pair;

[0066] (4) Using ① and ② as templates, select Uarm-F / Darm-R primer pair to amplify up-down fragments, also known as Donor DNA.

[0067] 2. Preparation and electroporation of competent cells

[0068] (1) 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) was electroporated into MHZ-0215-2 competent cells (the transformation method and the preparation method of competent cells are both referred to Molecular Cloning III);

[0069] (2) Pick a single colony of MHZ-0215-2 (pCas) into a 5 mL LB tube containing kanamycin and 10 mM arabinose, and incubate at 30 °C and 200 r / min until OD. 650 Electrocompetent cells were prepared after 0.8 (the method for preparing competent cells is described in Molecular Cloning III).

[0070] (3) The pTargetF-N20(ΔynaI) plasmid and Donor DNA were simultaneously electroporated into MHZ-0215-2(pCas) competent cells (electroplation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30℃ until single colonies were visible.

[0071] 3. Recombination Verification

[0072] (1) Use primer pair ynaI-F / ynaI-R to perform colony PCR amplification on the above single colonies;

[0073] (2) The amplified products were sent for sequencing to verify the integrity of the sequence.

[0074] 4. Construct recombinant bacteria with loss of relevant plasmids

[0075] (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 it onto an LB plate containing kanamycin.

[0076] (2) Pick a single colony and spot it onto LB plates containing kanamycin, spectinomycin and kanamycin alone, and incubate overnight at 30°C. If it cannot grow on LB plates containing kanamycin or spectinomycin, but grows on LB plates containing kanamycin, it indicates that the pTargetF-N20(ΔynaI) plasmid has been lost.

[0077] (3) Pick positive colonies that have lost pTargetF-N20(ΔynaI) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them on LB plates and incubate overnight at 37℃.

[0078] (4) Select a single colony and spot it on 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 strain MHZ-0221-18 is obtained.

[0079] The threonine-producing genetically modified strains obtained in Example 1 are shown in Table 2.

[0080] Table 2. Genetically engineered bacteria constructed in Example 1

[0081] strain number genotype 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

[0082] Example 2: Verification by shake-flask fermentation of L-threonine-producing genetically engineered bacteria

[0083] The recombinant strain MHZ-0221-18 constructed in Example 1 and its original strain MHZ-0215-2 were subjected to shake-flask fermentation verification for L-threonine production, as detailed below:

[0084] 1. Take two strains, MHZ-0215-2 and MHZ-0221-18, from the cryopreservation tubes, streak them on LB plates for activation, and incubate at 37°C for 24 hours;

[0085] 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 220 rpm for approximately 5 hours to allow the OD to reach its maximum. 650 Keep it below 1.7;

[0086] 3. Transfer 1 mL of seed culture to a shake flask containing 50 mL of fermentation medium (see Table 4), and ferment on a shaker at 37°C and 135 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, the shake flasks were repeated three times, and the average values ​​of the threonine yield and sugar-acid conversion rate are shown in Table 5.

[0087] Table 3 Seed Culture Medium

[0088] Element concentration glucose 30g / L Corn syrup 20g / L Soybean meal hydrolysate 5g / L Yeast paste 5g / L <![CDATA[KH2PO4]]> 2.5g / L Magnesium sulfate heptahydrate 0.7g / L <![CDATA[FeSO4、MnSO4]]> 20mg / L pH 7.0

[0089] Table 4 Fermentation Culture Media

[0090] Element concentration glucose 70g / L Corn syrup 10g / L Soybean meal hydrolysate 10g / L Magnesium sulfate heptahydrate 1.5g / L <![CDATA[KH2PO4]]> 1.5g / L Aspartic acid 15g / L <![CDATA[FeSO4]]> 30mg / L <![CDATA[MnSO4]]> 30mg / L Thiamine 500μg pH 7.0

[0091] Table 5. Comparison of productivity of threonine-producing genetically engineered bacteria

[0092]

[0093] Table 5 shows that the L-threonine production of the novel *E. coli* strain MHZ-0221-18 described in this invention is higher than that of the control strain MHZ-0215-2. The average conversion rate of the modified strain MHZ-0221-18 during shake-flask fermentation was 18.26%, which is 2.05 percentage points higher than that of the original strain. These shake-flask fermentation results indicate that the threonine production capacity of the modified strain MHZ-0221-18 is significantly better than that of the original strain MHZ-0215-2. Therefore, the knockout of the ynaI gene can significantly improve the threonine production capacity of the strain.

[0094] 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. A recombinant Escherichia coli, characterized in that, The recombinant E. coli was obtained by inactivating the mechanosensitive protein YnaI in E. coli MHZ-0215-2. The Escherichia coli MHZ-0215-2 has the accession number CGMCC No. 13403; The amino acid sequence of the mechanosensitive protein YnaI is shown in SEQ ID NO.1; The recombinant Escherichia coli exhibits increased L-threonine production due to the inactivation of the mechanosensitive protein YnaI.

2. The method for constructing recombinant Escherichia coli according to claim 1, characterized in that, include: The mechanosensitive protein YnaI in Escherichia coli MHZ-0215-2 was inactivated using genetic engineering methods.

3. The application of the recombinant Escherichia coli according to claim 1 in the production of L-threonine.

4. The application of the recombinant Escherichia coli of claim 1 or the construction method of claim 2 in the selection of L-threonine producing strains.

5. A method for producing L-threonine by fermentation, characterized in that, L-threonine was obtained by fermentation using the recombinant Escherichia coli according to claim 1.

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