Recombinant strains and methods for producing amino acids

Abstract

The present application relates to the field of microbial technology, in particular to a recombinant strain and a method for producing amino acid. The present application provides a recombinant strain, compared with the starting strain, the recombinant strain comprises: the expression and / or enzyme activity of global regulator FNR or its homolog or its functional variant is reduced. The present application significantly improves the yield and conversion rate of threonine and other amino acids by reducing the expression of global regulator FNR in the strain. The yield and conversion rate of threonine of the recombinant strain provided by the present application are significantly improved compared with the starting strain, which is beneficial to reduce the fermentation production cost of threonine, and provides an effective modification target and strain for the breeding of high-yield threonine strains.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to recombinant strains and methods for producing amino acids. Background Technology

[0002] Currently, amino acids are widely used in food, feed, cosmetics, health products, pharmaceuticals, and chemical industries. The main methods for producing amino acids include microbial fermentation, chemical synthesis, and enzymatic methods. Among these, microbial fermentation has become the primary method for amino acid production due to its numerous advantages.

[0003] L-Threonine is one of the essential amino acids for the human body. Currently, the production of L-Threonine using various bacteria has been reported, such as mutant strains obtained through mutagenesis of wild-type strains of *Escherichia coli*, *Corynebacterium*, and *Serratia*, including amino acid analogue-resistant mutants or auxotrophic strains containing methionine, lysine, isoleucine, etc. (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. With the development of genetic engineering technology, there are reports of constructing threonine-producing strains using genetic engineering techniques. Chinese Patent CN03811059.8 discloses the use of *Escherichia coli*, by deleting a 39bp sequence from position -56 to -18 of the threonine operon sequence, to enhance the expression of the key gene *thrABC* for threonine synthesis, resulting in a 22% increase in threonine productivity. Kwang Ho Lee et al. (Systemsmetabolic engineering of Escherichia coli for L-threonine production, Mol SystBiol. 2007; 3:149) utilized 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 inactivating the competing 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 hours of fermentation, with a sugar-acid conversion rate of 39.3%. Chinese patent 201611250306.8 discloses the MHZ-0215-2 strain obtained by strengthening the pntAB gene and heterologously introducing the pyc gene. This strain produced 12.4 g / L of threonine with a conversion rate of approximately 16.2%, and this strain had no plasmid burden.

[0004] The fermentation performance of amino acid production strains is a key factor determining amino acid yield and conversion rate. Therefore, improving the fermentation performance of production strains is of great significance for increasing amino acid yield and conversion rate and further reducing costs. 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 strain, which, compared with its originating strain, comprises: reduced expression and / or reduced enzyme activity of the global regulatory protein FNR or its homologs or functional variants.

[0008] The global regulatory protein FNR is a homodimeric DNA-binding protein containing an oxygen-labile iron-sulfur center that acts as an anaerobic sensor. Each subunit of the FNR protein contains approximately one [4Fe-4S] core. 2+ The unit, FNR, as a transcription factor, depends on [4Fe-4S]. 2+ The integrity of the unit is crucial because it promotes the dimerization of FNR. The oxygen sensitivity of the FNR protein is also influenced by [4Fe-4S]. 2+ The [4Fe-4S] mediated in the presence of O2 2+ It will transform into [2Fe-2S] 2+ FNR's [2Fe-2S] 2+ The structure exists as a monomer in solution and does not participate in DNA binding or transcription.

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

[0010] In this invention, the global regulatory protein FNR has the amino acid sequence shown in SEQ ID NO.1, or has the amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acids from the amino acid sequence shown in SEQ ID NO.1, or has an amino acid sequence with at least 80% homology to the amino acid sequence shown in SEQ ID NO.1.

[0011] In Escherichia coli, the amino acid sequence of the global regulatory protein FNR is shown in SEQ ID NO.1, and the ID number of its encoding gene fnr is 945908, and its nucleotide sequence is shown in SEQ ID NO.2.

[0012] 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):

[0013] (1) Insert, delete or replace one or more bases in the gene encoding the global regulatory protein FNR to reduce the expression level of the global regulatory protein FNR, reduce enzyme activity or inactivate it.

[0014] (2) Replace the transcriptional or translational regulatory element of the gene encoding the global regulatory protein FNR with a less active regulatory element so as to reduce its expression, reduce enzyme activity or inactivate it.

[0015] In a preferred embodiment of the present invention, the expression and / or enzyme activity of the global regulatory protein FNR are reduced by inactivating the protein.

[0016] 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.

[0017] Specifically, the starting strain preferably contains one or more of the following mutations:

[0018] (1) Enhanced expression of the pntAB gene;

[0019] (2) Expression of the pyc gene derived from Corynebacterium glutamicum;

[0020] (3) The mutant thrA*(S345P) expressing thrA;

[0021] (4) Knock out the tdh gene;

[0022] (5) Increase the number of copies of thrA*(S345P)BC.

[0023] 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.

[0024] Decreased expression and / or enzyme activity of the global regulatory protein FNR can significantly increase the production of threonine, glycine, or isoleucine. Therefore, the amino acid used in this invention is preferably threonine, glycine, or isoleucine.

[0025] The recombinant strains described in this invention are bacteria selected from the genera Escherichia, Corynebacterium, and Serratia.

[0026] 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.

[0027] Preferably, the recombinant strain is Escherichia coli.

[0028] The present invention also provides a method for constructing the recombinant microorganism, the method comprising: reducing the expression and / or enzyme activity of the global regulatory protein FNR in the starting strain by means of genetic engineering or mutagenesis.

[0029] 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.

[0030] In one embodiment of the present invention, the genetic engineering method is CRISPR / Cas9 technology. Specifically, a plasmid containing sgRNA targeting the fnr gene, upstream and downstream homologous arms of the fnr gene, and Cas9 protein is introduced into the starting strain, and the fnr gene is knocked out through homologous recombination.

[0031] The recombinant strains provided by this invention significantly improve the yield and conversion rate of amino acids (especially threonine) or their derivatives.

[0032] Based on this, the present invention provides the application of the recombinant strain in the production of amino acids or their derivatives.

[0033] The present invention also provides the application of the recombinant strain or its construction method in the selection of amino acid production strains.

[0034] In the applications described above, preferably, the amino acid is threonine, glycine, or isoleucine. More preferably, it is threonine.

[0035] Based on the novel function of the global regulatory protein FNR, the present invention also provides any of the following applications of the global regulatory protein FNR or its repressor, the encoding gene of the global regulatory protein FNR or its repressor, and biological materials containing said encoding gene or said repressor:

[0036] (1) Application in increasing the yield and / or conversion rate of amino acids or their derivatives in microorganisms;

[0037] (2) Application in the construction of production strains for amino acids or their derivatives;

[0038] (3) Application in the fermentation production of amino acids or their derivatives;

[0039] Preferably, the application is achieved by reducing the expression and / or enzyme activity of the global regulatory protein FNR.

[0040] The inhibitors mentioned above are proteins, DNA, or RNA that can inhibit the expression and / or enzyme activity of the global regulatory protein FNR.

[0041] The biological materials mentioned above include recombinant DNA, expression cassettes, vectors, or microorganisms.

[0042] The present invention also provides a method for increasing the amino acid production of a strain, comprising: reducing the expression and / or enzyme activity of the global regulatory protein FNR of the strain.

[0043] 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 strain and recovering the amino acids or their derivatives from the obtained culture medium.

[0044] Preferably, the amino acid is threonine, glycine, or isoleucine. More preferably, it is threonine.

[0045] For threonine, the fermentation medium used to culture the recombinant strain 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.

[0046] For threonine, the seed culture medium used to cultivate the recombinant strain 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.

[0047] The above-described method for fermenting to produce amino acids or their derivatives includes: first, culturing an activated recombinant strain 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.

[0048] The beneficial effects of this invention are as follows: By reducing the expression of the global regulatory protein Fnr in the starting strain, this invention significantly improves the yield and conversion rate of amino acids such as threonine. The recombinant strain with reduced expression of the global regulatory protein Fnr provided by this invention significantly improves the threonine yield and conversion rate compared to 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

[0049] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0050] 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 coding gene for FNR, specifically by knocking out the gene fnr, Gene ID: 945908.

[0051] 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).

[0052] 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.

[0053] All reagents used in the following examples are commercially available.

[0054] The primer sequences used in the following examples are shown in Table 1.

[0055] Table 1 Primer sequences used in the examples

[0056]

[0057] The present invention will be further illustrated below with reference to the embodiments.

[0058] Example 1: Preparation of fnr gene knockout strain MHZ-0221-5

[0059] 1. Construction of pTargetF-N20(Δfnr) plasmid and Donor DNA

[0060] (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 with 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(Δfnr), which was then identified by PCR and sequenced for verification.

[0061] (2) Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the Uarm-F / Uarm-R primer pair;

[0062] (3) Using the W3110 genome as a template, the downstream homologous arm ② was amplified by using the Darm-F / Darm-R primer pair;

[0063] (4) Using ① and ② as templates, select Uarm-F / Darm-R primer pair to amplify up-down fragments, also known as Donor DNA.

[0064] 2. Preparation and electroporation of competent cells

[0065] (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);

[0066] (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 0.4% concentration (the method for preparing competent cells is described in Molecular Cloning III).

[0067] (3) Electroporate pTargetF-N20(Δfnr) plasmid and Donor DNA into MHZ-0215-2(pCas) competent cells simultaneously (electroplation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30℃ until single colonies are visible.

[0068] 3. Recombination Verification

[0069] (1) Use primer pair fnr-F / fnr-R to perform colony PCR amplification on the above single colonies;

[0070] (2) The amplified products were sent for sequencing to verify the integrity of the sequence.

[0071] 4. Construct recombinant bacteria with loss of relevant plasmids

[0072] (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.

[0073] (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 and spectinomycin, but grows on LB plates containing kanamycin, it indicates that the pTargetF-N20(Δfnr) plasmid has been lost.

[0074] (3) Pick positive colonies that have lost pTargetF-N20(Δfnr) 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℃.

[0075] (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-0221-5 is obtained.

[0076] The threonine-producing genetically modified strains obtained in Example 1 are shown in Table 2.

[0077] Table 2 Genetically engineered bacteria constructed in Example 1

[0078] strain number genotype MHZ-0215-2 W3110(thrA*(S345P),tdh::thrA*BC,Ptac-pntAB,IS4::P1-pyc) MHZ-0221-5 W3110(thrA*(S345P),tdh::thrA*BC,Ptac-pntAB,IS4::P1-pyc,Δfnr

[0079] Example 2: Verification by shake-flask fermentation of L-threonine-producing genetically engineered bacteria

[0080] The recombinant strain MHZ-0221-5 constructed in Example 1 and its originating strain MHZ-0215-2 were subjected to shake-flask fermentation verification for L-threonine production, as detailed below:

[0081] 1. Take two strains, MHZ-0215-2 and MHZ-0221-5, from the cryopreservation tubes, streak them on LB plates for activation, and incubate at 37°C for 24 hours;

[0082] 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 1.8;

[0083] 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.

[0084] Table 3 Seed Culture Medium

[0085] 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

[0086] Table 4 Fermentation Culture Media

[0087] 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

[0088] Table 5. Comparison of productivity of threonine-producing genetically engineered bacteria

[0089]

[0090] Table 5 shows that the L-threonine production of the novel *E. coli* strain MHZ-0221-5 described in this invention is higher than that of the control strain MHZ-0215-2. The average conversion rate of the modified strain MHZ-0221-5 during shake-flask fermentation was 18.31%, which is 2.1 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-0221-5 is significantly better than that of the original strain MHZ-0215-2. Therefore, knocking out the *fnr* gene can significantly improve the threonine production capacity of the strain.

[0091] 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. sequence list <110> Li Yan <120> Recombinant strains and methods for producing amino acids <130> KHP211117781.9 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 250 <212> PRT <213> Artificial Sequence <400> 1 Met Ile Pro Glu Lys Arg Ile Ile Arg Arg Ile Gln Ser Gly Gly Cys 1 5 10 15 Ala Ile His Cys Gln Asp Cys Ser Ile Ser Gln Leu Cys Ile Pro Phe 20 25 30 Thr Leu Asn Glu His Glu Leu Asp Gln Leu Asp Asn Ile Ile Glu Arg 35 40 45 Lys Lys Pro Ile Gln Lys Gly Gln Thr Leu Phe Lys Ala Gly Asp Glu 50 55 60 Leu Lys Ser Leu Tyr Ala Ile Arg Ser Gly Thr Ile Lys Ser Tyr Thr 65 70 75 80 Ile Thr Glu Gln Gly Asp Glu Gln Ile Thr Gly Phe His Leu Ala Gly 85 90 95 Asp Leu Val Gly Phe Asp Ala Ile Gly Ser Gly His His Pro Ser Phe 100 105 110 Ala Gln Ala Leu Glu Thr Ser Met Val Cys Glu Ile Pro Phe Glu Thr 115 120 125 Leu Asp Asp Leu Ser Gly Lys Met Pro Asn Leu Arg Gln Gln Met Met 130 135 140 Arg Leu Met Ser Gly Glu Ile Lys Gly Asp Gln Asp Met Ile Leu Leu 145 150 155 160 Leu Ser Lys Lys Asn Ala Glu Glu Arg Leu Ala Ala Phe Ile Tyr Asn 165 170 175 Leu Ser Arg Arg Phe Ala Gln Arg Gly Phe Ser Pro Arg Glu Phe Arg 180 185 190 Leu Thr Met Thr Arg Gly Asp Ile Gly Asn Tyr Leu Gly Leu Thr Val 195 200 205 Glu Thr Ile Ser Arg Leu Leu Gly Arg Phe Gln Lys Ser Gly Met Leu 210 215 220 Ala Val Lys Gly Lys Tyr Ile Thr Ile Glu Asn Asn Asp Ala Leu Ala 225 230 235 240 Gln Leu Ala Gly His Thr Arg Asn Val Ala 245 250 <210> 2 <211> 753 <212> DNA <213> Artificial Sequence <400> 2 atgatcccgg aaaagcgaat tatacggcgc attcagtctg gcggttgtgc tatccattgc 60 caggattgca gcatcagcca gctttgcatc ccgttcacac tcaacgaaca tgagcttgat 120 cagcttgata atatcattga gcggaagaag cctattcaga aaggccagac gctgtttaag 180 gctggtgatg aacttaaatc gctttatgcc atccgctccg gtacgattaa aagttatacc 240 atcactgagc aaggcgacga gcaaatcact ggtttccatt tagcaggcga cctggtggga 300 tttgacgcca tcggcagcgg ccatcacccg agcttcgcgc aggcgctgga aacctcgatg 360 gtatgtgaaa tcccgttcga aacgctggac gattgtccg gtaaaatgcc gaatctgcgt cagcagatga tgcgtctgat gagcggtga atcaaaggcg atcaggacat gatcctgctg ttgtcgaaga aaaatgccga ggaacgtctg gctgcattca tctacaacct gtcccgtcgt 540 tttgcccaac gcggcttctc ccctcgtgaa ttccgcctga cgatgactcg tggcgatatc 600 ggtaactatc tgggcctgac ggtagaaacc atcagccgtc tgctgggtcg cttccagaaa agcggcatgc tggcagtcaa aggtaaatac atcaccatcg aaaataacga tgcgctggcc cagcttgctg gtcatacgcg taacgttgcc tga <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <400> 3 45 years old gcattcagtc tgggttttag <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <400> 4 ccagactgaa tgcgccgtat actagtatta tacctaggac tgagct <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <400> 5 ccggtaatct tagcattatt g 21 <210> 6 <211> 50 <212> DNA <213> Artificial Sequence <400> 6 aaggatagtg agttatgcgg aaaaaaggtc tgctcaagcc gtaattgata 50 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <400> 7 tatcaattac ggcttgagca gacctttttt ccgcataact cactatcctt 50 <210> 8 <211> twenty one <212> DNA <213> Artificial Sequence <400> 8 cgccattttt agcaccaaat c 21 <210> 9 <211> twenty one <212> DNA <213> Artificial Sequence <400> 9 ctgctggaca tccattatcg c 21 <210> 10 <211> twenty one <212> DNA <213> Artificial Sequence <400> 10 ggtctttcac catccacacc g 21

Claims

1. Use of an inactivated global regulator protein FNR to increase the yield and / or conversion of threonine in Escherichia coli; wherein, The amino acid sequence of the global regulator FNR is shown as SEQ ID NO.

1.

2. Use of an inactivated global regulator protein FNR in the construction of a strain for the production of threonine; wherein, The amino acid sequence of the global regulator FNR is shown as SEQ ID NO. 1; and the production strain is Escherichia coli.

3. Use of an inactivated global regulator protein FNR in the fermentative production of threonine; wherein, The amino acid sequence of the global regulator FNR is shown as SEQ ID NO. 1, and the production strain used in the fermentation production is Escherichia coli.

4. Application of recombinant Escherichia coli in threonine production; wherein The mutation of the recombinant Escherichia coli compared with the starting strain comprises inactivation of the global regulator FNR, wherein the amino acid sequence of the global regulator FNR is shown as SEQ ID NO. 1; The starting strain is a strain capable of accumulating threonine.

5. Use according to claim 4, characterized in that, The starting strain contains one or more of the following mutations: (1) enhanced expression of pntAB gene; (2) expression of pyc gene derived from Corynebacterium glutamicum; (3) a mutant thrA expressing thrA S345P ; (4) knockout of tdh gene; (5) increase thrA S345P Copy number of BC.

6. Application of recombinant Escherichia coli in selection of threonine production strain; wherein The mutation of the recombinant Escherichia coli compared with the starting strain comprises inactivation of the global regulator FNR, wherein the amino acid sequence of the global regulator FNR is shown as SEQ ID NO. 1; The starting strain is a strain capable of accumulating threonine.

7. Use according to claim 6, characterized in that, The starting strain contains one or more of the following mutations: (1) enhanced expression of pntAB gene; (2) expression of pyc gene derived from Corynebacterium glutamicum; (3) a mutant thrA expressing thrA S345P ; (4) knockout of tdh gene; (5) increase thrA S345P copy number of BC.

8. A method for the fermentative production of threonine, characterized in that Culturing the recombinant Escherichia coli, and recovering threonine from the obtained culture solution; The mutation of the recombinant Escherichia coli compared with the starting strain comprises inactivation of the global regulator FNR, wherein the amino acid sequence of the global regulator FNR is shown as SEQ ID NO. 1; The starting strain is a strain capable of accumulating threonine.

9. The method of claim 8, wherein, The starting strain contains one or more of the following mutations: (1) enhanced expression of pntAB gene; (2) expression of pyc gene derived from Corynebacterium glutamicum; (3) a mutant thrA expressing thrA S345P ; (4) knockout of tdh gene; (5) increase thrA S345P Copy number of BC.

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