Mutant YbiI protein as well as gene, recombinant strain, application and production method thereof

Abstract

The invention belongs to the field of microbial fermentation, and particularly relates to a mutant YbiI protein and a gene, a recombinant strain, application and a production method thereof. According to the mutant YbiI protein provided by the invention, relative to a wild YbiI protein with a sequence as shown in SEQ ID NO: 1, the mutant YbiI protein at least comprises the following mutation sites: the 86th amino acid sequence of a wild ybiI gene coding protein is mutated into leucine from glutamine. The gene of the mutant YbiI protein is introduced into other strains to replace a wild type ybiI gene, the threonine yield of different strains can be increased, when a recombinant THRS strain is in a 5L fermentation tank, the threonine yield is increased by 7.47%, and the threonine yield reaches 107.8 g / L after 48-hour fermentation. When the recombinant THRS-6 strain is placed in a 5L fermentation tank, the threonine yield is improved by 13.41%, and the threonine yield reaches 142.1 g / L after 48 hours of fermentation.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and specifically relates to a mutant YbiI protein and a gene, a recombinant strain, an application and a production method thereof. Background Art

[0002] Threonine is one of the eight amino acids for human and animal growth and is widely used in medicine, chemical reagents, food fortifiers, feed additives, etc. Threonine is an important nutritional fortifier that can be used in cereals, pastries, and dairy products. Like tryptophan, it can relieve human fatigue and promote growth and development. In medicine, because threonine contains hydroxyl groups in its structure, it has a water-retaining effect on human skin. It combines with oligosaccharide chains and plays an important role in protecting cell membranes. It can promote phospholipid synthesis and fatty acid oxidation in the body. At present, threonine is mainly produced industrially by microbial fermentation. Microorganisms are usually modified to increase the production of L-threonine. A variety of bacteria can be used in the production of L-threonine, such as wild-type induced mutants of Escherichia, Corynebacterium, and Brevibacterium as production strains. With the increasing global demand for threonine, the construction and transformation of high-yield threonine strains are particularly important.

[0003] At present, the production and conversion rate of amino acids are mainly increased by modifying some key genes in the amino acid metabolic pathway. Some genes in key positions can control the expression level of enzymes and the anti-feedback ability of gene transcription. For example, CN110592084A uses Escherichia coli K12 or its derivative strain W3110CGMCC 7.232 to introduce point mutations rhtA gene, mutating its 67th base from adenine (A) to guanine (G), thereby improving the recombinant strain's ability to produce threonine; Jianli Wang et al. overexpressed a gene cluster from Ralstonia eutropha phaCAB , the Escherichia coli strain TWF001 was modified to increase the fermentation yield of L-threonine; Jun Yang et al. effectively increased the threonine production by strengthening fatty acid degradation, weakening fatty acid synthesis, and accelerating the glyoxylate cycle.

[0004] However, there are relatively few studies on the modification of genes related to the uptake of important nutrients in the environment to increase the production and conversion rate of amino acids. In Gram-negative bacteria, important nutrients in the environment are mainly taken up through the TonB system (i.e., TBDT). TonB-dependent receptors exist in all Gram-negative bacteria. Studies by Yaling Wang et al. have shown that TonB-dependent receptors are not only involved in the uptake of transition metal elements such as iron, manganese, zinc, and nickel, as well as a variety of nutrients such as vitamins, heme, and carbohydrates, but also in the secretion of proteases. Transition metals, as metal cofactors, play an important role in biological metabolism, including precursor biosynthesis, DNA replication, transcription, respiration, and oxidative stress response. Escherichia coli is a Gram-negative bacterium. In Escherichia coli, the genes encoding TBDT are scattered throughout the chromosome, for example btuB、fecA、ybiX and ybiI The expression of these genes is highly regulated at both the transcriptional and post-transcriptional levels. ybiI The gene contains zinc finger domain protein. The function of this structure is applicable to the local structural form of protein molecules that bind or match each other between biological macromolecules. It is one of the structural guarantee factors for cooperating with the gene transcription and regulation of organisms. Summary of the invention

[0005] In order to solve the above technical problems, the present invention performs whole genome sequencing on a high-threonine-producing mutant bacterium FFTHR-1 in the laboratory, and finds that there is a mutation at position 86 in the YbiI protein, which is one of the reasons for the high threonine production of the mutant bacterium; introducing its encoding gene into other production strains can also increase the threonine production.

[0006] The first aspect of the present invention provides a mutant YbiI protein. Compared with the wild-type YbiI protein whose sequence is shown in SEQ ID NO: 1, the mutant YbiI protein comprises at least the following mutation site: the 86th amino acid of the wild-type YbiI protein is mutated from glutamine to leucine.

[0007] In some embodiments, the mutant YbiI protein may also contain other amino acid substitutions, deletions, insertions, additions or inversions, as long as the mutation does not affect the function of the YbiI protein. In a specific embodiment, the amino acid sequence of the mutant YbiI protein is shown in SEQ ID NO:2.

[0008] The present invention also provides a mutant ybiI gene, the mutant ybiI The gene encodes the mutant YbiI protein. ybiI The gene can be wild type ybiIThe gene sequence is obtained by gene mutation or designed according to the amino acid sequence.

[0009] In some embodiments, relative to the wild-type sequence shown in SEQ ID NO: 3 ybiI gene, the mutant ybiI The gene contains at least the following mutation sites: wild type ybiI The 257th base of the gene mutated from T to A.

[0010] In some embodiments, the mutant ybiI The gene may also contain other one or more base mutations, such as codon optimization mutations, conservative sequence mutations, etc. In a specific embodiment, the mutant ybiI The nucleotide sequence of the gene is shown in SEQ ID NO: 4. The present invention also provides the mutant YbiI protein or the mutant ybiI Application of genes to improve threonine production in threonine-producing strains.

[0011] The present invention also provides a recombinant strain for high-yield L-threonine, wherein the recombinant strain expresses the mutant YbiI protein, or the recombinant strain ybiI The gene is replaced by the mutant ybiI Gene.

[0012] In some embodiments, the recombinant strain ybiI The gene is replaced with the mutant by mutagenesis, PCR site-directed mutagenesis or homologous recombination. ybiI Gene.

[0013] In some embodiments, the recombinant strain is Escherichia coli, preferably based on Escherichia coli THRS or Escherichia coli THRS-6. The Escherichia coli THRS is the Escherichia coli THRS strain in patent CN202411834458.7, which was deposited in the China General Microbiological Culture Collection Management Center on August 20, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.31694, and a classification name of Escherichia coli Escherichiacoli. The Escherichia coli THRS-6 is the Escherichia coli THRS-6 strain in patent CN202411834455.3, which was deposited in the China General Microbiological Culture Collection Management Center on August 20, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.31695, and a classification name of Escherichia coli Escherichia coli.

[0014] The present invention also provides a method for preparing the recombinant strain, comprising the following steps: ybiI The homologous arm primers PybiI-F / PybiI-R were designed using the gene as a template to construct PCR amplification products, which were transformed into threonine production strains, and the target region was replaced by homologous recombination to obtain a recombinant strain.

[0015] The present invention also provides a method for producing L-threonine, comprising the following steps: inoculating a recombinant strain on a seed culture medium for culturing to obtain a seed solution, and transferring the seed solution into a fermentation culture medium at an inoculation rate of 20% to produce L-threonine.

[0016] In some embodiments, the seed culture medium is: 5 g / L corn steep liquor powder, 20 g / L glucose, 5 g / L yeast powder, 2 g / L KH2PO4, 1 g / L magnesium sulfate, 20 mg / L FeSO4·7H2O, and 20 mg / L MnSO4·H2O; the fermentation medium is: 20 g / L glucose, 2 g / L potassium dihydrogen phosphate, 3 g / L yeast powder, 1 g / L betaine, 1 g / L magnesium sulfate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 8 g / L corn steep liquor powder, and 10 mg / L vitamin B1.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least: The present invention provides a mutant YbiI protein, which can be expressed in strains to increase the threonine production of different strains. For example, the threonine production of the recombinant THRS strain in a 5L fermenter is increased by 7.47%, and the threonine production after 48h fermentation reaches 107.8g / L; the threonine production of the recombinant THRS-6 strain in a 5L fermenter is increased by 13.41%, and the threonine production after 48h fermentation reaches 142.1g / L. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0019] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0020] Example 1: Construction of recombinant strains using THRS strains The THRS strain was transformed into ybiIGene knockout and knock-in, the specific steps are as follows: Prepare competent E. coli cells, use arabinose stock solution to induce pKD46 plasmid into competent E. coli cells, and culture and screen in a resistance medium containing ampicillin. Select homology arms at both ends of the target gene, and design kanamycin resistance gene using plasmid PKD13 as a template can The homologous arm primers Pkan-F / Pkan-R were used for PCR amplification, and the purified PCR product was transferred into the strain containing the pKD46 plasmid by electroporation to obtain the pKD46-kan strain. Homologous recombination occurred during the culture at 30°C to obtain the homologous recombination strain. The pKD46 temperature-sensitive plasmid was removed by culture at 37°C. Resistance screening was performed on a medium containing kanamycin to obtain the strain with the target gene removed. PCR was performed with the primer pair PybiI-F' / PybiI-R' to verify whether the target gene was successfully removed. Subsequently, the pCP20 plasmid was introduced and transformed to express the flippase recombinase gene to promote the homologous recombination of the FRT site itself, and finally achieve ybiI Knockout of genes. ybiI The knockout strains were cultured in LB medium and kanamycin resistance medium at the same time. The strains that grew normally in LB medium but did not grow in resistance medium were ybiI The knockout strain was cultured at 42°C to remove the pCP20 temperature-sensitive plasmid and verified by PCR amplification with the primer pair PpCP20-F / PpCP20-R. ybiI The knockout strain THRS-△ybiI.

[0021] Mutant ybiI Gene sequence (SEQ ID NO: 4, named ybiI T257A ) was used as a template to design homology arm primers PybiI-F / PybiI-R and construct exogenous ybiI T257A The PCR amplification product of the gene is transformed into Escherichia coli THRS, and the target region is replaced by homologous recombination. The specific implementation method is the same as above, and finally the ybiI T257A The knock-in of the mutant gene, PCR verification and sequencing analysis were performed on the above recombinant strain, and the sequencing result was shown as SEQ ID NO: 4, that is, ybiI T257A Mutant strain THRS-YbiI Q86L.

[0022] Example 2: Fermentation of recombinant strains to produce L-threonine The obtained in Example 1 ybiI T257A Mutant strains, ybiIThe knockout strain and the wild-type THRS strain were inoculated on a seed medium to obtain a seed solution; the seed medium included the following components: corn steep liquor 4.2 g / L, glucose 10 g / L, yeast powder 2.5 g / L, KH2PO4 2g / L, magnesium sulfate 1.2 g / L, FeSO4·7H2O 20 mg / L and MnSO4·H2O 20 mg / L, biotin 30 mg / L. Three parallel experiments were set up for each group of experiments.

[0023] The seed culture obtained by the above-mentioned culture method is transferred into the fermentation medium at a 20% inoculation rate for culture: 2.1. 5L seed tank process control a) Adjust the temperature to 37°C, pH to 7.0, speed to 500 rpm, and air volume to 0.3 m 3 / h, the whole process temperature was controlled at 37℃, the tank pressure was 0.05~0.08 MPa, and the culture period was 10 h; b) Transplantation standard: OD600: 12-15.

[0024] c) Seed culture medium is corn steep liquor powder 5 g / L, glucose 20 g / L, yeast powder 5 g / L, KH2PO4 2 g / L, magnesium sulfate 1 g / L, FeSO4·7H2O 20 mg / L, MnSO4·H2O 20 mg / L; 2.2, 5 L fermenter fermentation process control a) Adjust the temperature to 37°C, pH to 7.0, initial speed to 300 rpm, and air volume to 0.3 m 3 / h, the whole process temperature is controlled at 37℃, and the tank pressure is 0.05~0.08 MPa; b) DO control: at 0 h, air volume 0.3 m 3 / h, 300 rpm, tank pressure 0.05 MPa; c) When DO drops below 30%, the dissolved oxygen level is controlled at 30% by adjusting the ventilation volume and stirring speed until the fermentation ends; d) The fermentation medium was 20 g / L glucose, 2 g / L potassium dihydrogen phosphate, 3 g / L yeast powder, 1 g / L betaine, 1 g / L magnesium sulfate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 8 g / L corn steep liquor powder, and 10 mg / L vitamin B1.

[0025] 2.3, 5 L fermenter ybiI T257A Verification of strain production performance After determination, the L-threonine fermentation yields of different strains are shown in Table 1. The results show that, ybiIAfter the 86th glutamine in the amino acid sequence of the gene was replaced by leucine, the L-threonine production was significantly increased compared with the original strain. At the same time, the strain THRS-YbiI Q86L was named strain FFTHR-36.

[0026] Table 1: L-threonine production performance of strains in 5 L fermenter Example 3: Construction of a recombinant strain using THRS-6 strain and fermentation to produce L-threonine According to the method in Example 1, strain THRS-6-Δ was constructed based on THRS-6. ybiI and THRS-6-YbiIQ86L. The yields of the original strain and the modified strain were verified by fermentation in a 5 L fermenter, and the fermentation method was referred to Example 2. At the same time, the strain THRS-6-YbiI Q86L was named strain FFTHR-37. After measurement, the L-threonine fermentation yields of different strains are shown in Table 2. This shows that the mutant YbiI protein can increase the L-threonine fermentation yield in different strains.

[0027] Table 2: L-threonine production performance of strains in 5 L fermenter The sequence involved in the present invention is as follows: YbiI protein sequence: MASGWANDDAVNEQINSTIEDAIARARGEIPRGESLDECEECGAPIPQARREAIPGVRLCIHCQQEKDLQKPAYTGYNRRGSKDSQLR (SEQ ID NO:1) Mutant YbiI protein sequence: MASGWANDDAVNEQINSTIEDAIARARGEIPRGESLDECEECGAPIPQARREAIPGVRLCIHCQQEKDLQKPAYTGYNRRGSKDSLLR (SEQ ID NO: 2) ybiI Nucleotide sequence of the gene: ttaacgtaactggctgtctttcgaacctctgcgattatatcctgtataagctggtttttgtaaatctttctcctgctgacaatgaatacataagcgcacgccaggaatggcttcccgacgggcctgcgggatgggggcaccgcactcttcacattcatccaggctttcgccgcgcggaatttcaccccgagcgcgggcaatcgcatcttcaattgtactgttgatctgttcgttgacggcgtcatcgttagcccaaccggatgccat (SEQ ID NO:3).

[0028] Mutant ybiI Nucleotide sequence of the gene: ttaacgtaacaggctgtctttcgaacctctgcgattatatcctgtataagctggtttttgtaaatctttctcctgctgacaatgaatacataagcgcacgccaggaatggcttcccgacgggcctgcgggatgggggcaccgcactcttcacattcatccaggctttcgccgcgcggaatttcaccccgagcgcgggcaatcgcatcttcaattgtactgttgatctgttcgttgacggcgtcatcgttagcccaaccggatgccat (SEQ ID NO:4) Pkan-F: 5'-AAATACAAATTATACTCGCTATGCATCTCGCCAGTTGTAGAGGCTGGAGCTGCTTC-3' (SEQIDNO:5) Pkan-R: 5'-CTACACTTAACTGTACAAGTATTGATATGGGGAGGTCGATTCCGGGGATCCGTCGACC-3' (SEQIDNO:6) PybiI-F’: 5'-TTAACGTAACTGGCTGTCTTTCGAACCTC-3' (SEQIDNO:7) PybiI-R’: 5'-ATGGCATCCGGTTGGGCTAAC-3' (SEQIDNO:8) PpCP20-F: 5'-ATGTCTGAATTAGTTGTTTTCAAAGCAAATGAAC-3' (SEQ ID NO: 9) PpCP20-R: 5'-GATCCTCCGTATTTAGCCAGTATGTTCT-3' (SEQ ID NO: 10) PybI-F: 5'-AAATACAAATTATACTCGCTATGCATCTCGCCAGTTGTAGTTAACGTAACAGGCTGTCTTTCGAACCTC-3' (SEQ ID NO: 11) PybiI-R: 5'-CTACACTTAACTGTACAAGTATTGATATGGGGAGGTCGATATGGCATCCGGTTGGGCTAAC-3' (SEQ ID NO: 12) Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A mutant YbiI protein, characterized in that: Compared with the wild-type YbiI protein whose sequence is shown in SEQ ID NO: 1, the mutant YbiI protein at least comprises the following mutation site: the 86th amino acid of the wild-type YbiI protein is mutated from glutamine to leucine.

2. The mutant YbiI protein according to claim 1, characterized in that The amino acid sequence of the mutant YbiI protein is shown in SEQ ID NO:

2.

3. A mutant YbI A gene characterized by The mutant YbI The gene encodes the mutant YbiI protein according to claim 1 or 2.

4. The mutant according to claim 3 YbI A gene characterized by The mutant YbI The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

5. The mutant YbiI protein according to any one of claims 1 to 2, or the mutant YbiI protein according to any one of claims 3 to 4 YbI Application of genes to improve threonine production in threonine-producing strains.

6. A recombinant strain for high production of L-threonine, characterized in that: The recombinant strain expresses the mutant YbiI protein according to any one of claims 1 to 2, or the recombinant strain YbI The gene is replaced with a mutant according to any one of claims 3 to 4 YbI Gene.

7. The recombinant strain for high L-threonine production according to claim 5, characterized in that: The recombinant strain YbI The gene is replaced with the mutant by mutagenesis, PCR site-directed mutagenesis or homologous recombination. YbI Gene.

8. The recombinant strain for high L-threonine production according to claim 7, characterized in that: The recombinant strain is Escherichia coli.

9. The method for preparing a recombinant strain having a high yield of L-threonine according to any one of claims 6 to 8, characterized in that: The following steps are included: YbI The homology arm primers PybiI-F / PybiI-R were designed using the gene as a template to construct PCR amplification products, which were transformed into threonine production strains, and the target region was replaced by homologous recombination to obtain a recombinant strain.

10. A method for producing L-threonine, characterized in that: The method comprises the following steps: inoculating the high-yield L-threonine recombinant strain according to any one of claims 6 to 8 on a seed culture medium for cultivation to obtain a seed solution, and transferring the seed solution into a fermentation culture medium at an inoculation rate of 20% to produce L-threonine.

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