A mutant ybiI protein, gene thereof, recombinant strain, application and production method

Abstract

The application belongs to the field of microbial fermentation, and particularly relates to a mutant YbI protein, a gene thereof, a recombinant strain, application and a production method. The mutant YbI protein provided by the application comprises at least the following mutation site: the 86th amino acid sequence of the wild-type YbI protein coded by the wild-type gene is mutated from glutamine to leucine. ybiI The application introduces the gene of the mutant YbI protein into other strains to replace the wild-type gene, so that the threonine yield of different strains can be improved. ybiI The recombinant THRS strain can improve the threonine yield by 7.47% in a 5L fermenter, and the threonine yield reaches 107.8 g / L after 48h fermentation. The recombinant THRS-6 strain can improve the threonine yield by 13.41% in a 5L fermenter, and the threonine yield reaches 142.1 g / L after 48h fermentation.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, specifically relating to a mutant YbiI protein and its gene, recombinant strain, application and production method. Background Technology

[0002] Threonine is one of the eight essential amino acids for human and animal growth and is widely used in medicine, chemical reagents, food fortifiers, and feed additives. As an important nutritional fortifier, threonine can be used in grains, pastries, and dairy products, and like tryptophan, it can relieve fatigue and promote growth and development. In medicine, due to the hydroxyl group in its structure, threonine has a water-retaining effect on human skin. When combined with oligosaccharide chains, it plays an important role in protecting cell membranes and promotes phospholipid synthesis and fatty acid oxidation in vivo. Currently, threonine is mainly produced industrially through microbial fermentation. Microorganisms are often modified to increase L-threonine yield. Various bacteria can be used in L-threonine production, such as wild-type mutant strains induced from Escherichia coli, Corynebacterium, and Brevibacterium. With the increasing global demand for threonine, the construction and modification of high-yield threonine strains are particularly important.

[0003] Currently, increasing amino acid yield and conversion rate mainly involves modifying key genes in amino acid metabolism pathways. Some genes at key positions can control enzyme expression levels and the anti-feedback ability of gene transcription. For example, CN110592084A utilizes *Escherichia coli* K12 or its derivative strain W3110CGMCC 7.232 by introducing point mutations. rhtA The gene was modified by mutating the 67th base from adenine (A) to guanine (G), thereby enhancing the recombinant strain's ability to produce threonine; Jianli Wang et al. improved the recombinant strain's ability to produce threonine by overexpressing a gene cluster from the eutrophic algae Ralstonia eutropha. phaCAB Modifying the Escherichia coli strain TWF001 to increase the fermentation yield of L-threonine; Jun Yang et al. effectively increased threonine yield by enhancing fatty acid degradation and weakening fatty acid synthesis, and accelerating the glyoxylate cycle.

[0004] However, research on modifying genes related to the uptake of essential nutrients in the environment to improve amino acid production and conversion rates is relatively limited. In Gram-negative bacteria, the TonB system (TBDT) is primarily used for the uptake of essential nutrients. All Gram-negative bacteria possess TonB-dependent receptors. Studies by Yaling Wang et al. have shown that TonB-dependent receptors are involved not only in the uptake of transition metals such as iron, manganese, zinc, and nickel, as well as various nutrients such as vitamins, heme, and carbohydrates, but also in the secretion of proteases. Transition metals, as metal cofactors, play important roles in biological metabolism, including precursor biosynthesis, DNA replication, transcription, respiration, and oxidative stress responses. *Escherichia coli* is a Gram-negative bacterium. In *E. coli*, the gene encoding TBDT is distributed throughout the chromosome, for example... btuB、fecA、ybiX and ybiI Genes such as [list of genes] are highly regulated at both the transcriptional and post-transcriptional levels. ybiI Genes contain zinc finger domain proteins. The function of this structure is to serve as a local structural form on protein molecules that bind or anastomose between biological macromolecules. It is one of the structural guarantees for gene transcription and regulation in organisms. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention performed whole-genome sequencing on a high-threonine-producing mutant bacterium, FFTHR-1, which was found to have a mutation at position 86 of the YbiI protein, one of the reasons for the high threonine production of this mutant bacterium. Introducing its encoding gene into other producing strains can also increase the yield of threonine.

[0006] A first aspect of the present invention provides a mutant YbiI protein, which, relative to the wild-type YbiI protein with the sequence shown in SEQ ID NO: 1, includes 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 substitutions, deletions, insertions, additions, or inversions of other amino acids, as long as the mutation does not affect the function of the YbiI protein. In one 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 type ybiI Gene, the mutant ybiI The gene encodes the mutant YbiI protein. The mutant... ybiI Genes can be wild type ybiIGene sequences obtained through gene mutation or designed based on amino acid sequences.

[0009] In some implementations, relative to the wild type with a sequence such as 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 is mutated from T to A.

[0010] In some implementations, the mutant ybiI Genes may also contain mutations in one or more other bases, such as codon optimization mutations, conserved sequence mutations, etc. In one specific embodiment, the mutant... ybiI The nucleotide sequence of the gene is shown in SEQ ID NO: 4. This invention also provides the mutant YbiI protein or the mutant... ybiI Application of genes to increase threonine production in strains that produce threonine.

[0011] The present invention also provides a recombinant strain that produces high levels of L-threonine, wherein the recombinant strain expresses the mutant YbiI protein, or the protein of the recombinant strain. ybiI The gene was replaced with the mutant. ybiI Gene.

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

[0013] In some embodiments, the recombinant strain is *Escherichia coli*, preferably modified from *Escherichia coli THRS* or *Escherichia coli THRS-6*. The *Escherichia coli THRS* strain is the *Escherichia coli THRS* strain described in patent CN202411834458.7, which was deposited on August 20, 2024, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number CGMCC NO. 31694, and classified as *Escherichia coli*. The *Escherichia coli THRS-6* strain is the *Escherichia coli THRS-6* strain described in patent CN202411834455.3, which was also deposited on August 20, 2024, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number CGMCC NO. 31695, and classified as *Escherichia coli*.

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

[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 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 implementations, the seed culture medium consists of: 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 culture medium consists of: 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 following:

[0018] This invention provides a mutant YbiI protein, which, when expressed in a bacterial strain, can increase the threonine yield of different strains. For example, the recombinant THRS strain in a 5L fermenter increases the threonine yield by 7.47%, reaching 107.8 g / L after 48 hours of fermentation; the recombinant THRS-6 strain in a 5L fermenter increases the threonine yield by 13.41%, reaching 142.1 g / L after 48 hours of fermentation. Detailed Implementation

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in the following description are merely illustrative examples of specific implementations of this invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0020] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, which should be understood to include those close to them.

[0021] Example 1: Construction of recombinant strains using THRS strains

[0022] Homologous recombination was used to extract THRS strains ybiI The specific steps for gene knockout and knock-in are as follows:

[0023] Competent cells of *E. coli* were prepared. The pKD46 plasmid was transformed into competent *E. coli* cells using arabinose stock solution, and the cells were cultured and screened in an ampicillin-containing resistance medium. Homologous arms were selected from both ends of the target gene, and a kanamycin resistance gene was designed using plasmid PKD13 as a template. can PCR amplification was performed using the homologous arm primers Pkan-F / Pkan-R. The purified PCR product was then electroporated into a strain containing the pKD46 plasmid to obtain the pKD46-kan strain. Homologous recombination occurred during incubation at 30°C, yielding a recombinant strain. Incubation at 37°C removed the pKD46 temperature-sensitive plasmid. Anti-resistance selection was performed on kanamycin-containing medium to obtain strains with the target gene removed. PCR was performed using primer pair PybiI-F' / PybiI-R' to verify successful removal of the target gene. Subsequently, the pCP20 plasmid was introduced to express the flippant recombinase gene, promoting homologous recombination at the FRT site, ultimately achieving... ybiI Gene knockout. ybiI Gene knockout strains were cultured simultaneously in LB medium and kanamycin-resistant medium. Strains that grew normally in LB medium but not in the resistance medium were identified as gene knockout strains. ybiI Gene knockout strains were cultured at 42°C to remove the pCP20 temperature-sensitive plasmid. PCR amplification using the PpCP20-F / PpCP20-R primer pair was then performed to verify the result. ybiI The knockout strain THRS-△ybiI.

[0024] With mutant ybiI Gene sequence (SEQ ID NO: 4, named ybiI T257A Using this template, homologous arm primers PybiI-F / PybiI-R were designed to construct exogenous [products / materials]. ybiI T257A The PCR amplification product of the gene is transformed into E. coli THRS, and the target region is replaced by homologous recombination. The specific implementation method is the same as above, ultimately achieving... ybiI T257A The knock-in of the mutant gene was followed by PCR verification and sequencing analysis of the recombinant strain. The sequencing results are shown in SEQ ID NO:4, indicating that the recombinant strain was successfully obtained. ybiI T257A Mutant strain THRS-YbiI Q86L.

[0025] Example 2: Fermentation production of L-threonine by recombinant strains

[0026] The results obtained in Example 1 ybiI T257A mutant strains, ybiIGene knockout strains and wild-type THRS strains were inoculated onto seed culture medium to obtain seed solutions. The seed culture medium consisted of the following components at the following concentrations: corn steep liquor 4.2 g / L, glucose 10 g / L, yeast extract 2.5 g / L, KH₂PO₄ 2 g / L, magnesium sulfate 1.2 g / L, FeSO₄·7H₂O 20 mg / L and MnSO₄·H₂O 20 mg / L, and biotin 30 mg / L. Each experiment was conducted in triplicate.

[0027] The seed culture obtained using the above cultivation method was transferred into fermentation medium at an inoculum size of 20%.

[0028] 2.1 Process Control of 5L Seed Tank

[0029] a) Set the temperature to 37℃, pH to 7.0, fan speed to 500 rpm, and airflow to 0.3 m³. 3 / h, with the temperature controlled at 37℃ throughout the process, the tank pressure at 0.05~0.08 MPa, and the culture cycle at 10 h;

[0030] b) Transplanting standard: OD600: 12-15.

[0031] c) The seed culture medium consisted of 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.

[0032] 2.2 Fermentation process control of a 5 L fermenter

[0033] a) Set the temperature to 37℃, pH to 7.0, initial rotation speed to 300 rpm, and airflow to 0.3 m³ / h. 3 / h, with the temperature controlled at 37℃ throughout the process and the tank pressure at 0.05~0.08 MPa;

[0034] b) DO control: At 0 h, the air volume is 0.3 m³ / h. 3 / h, 300 rpm, tank pressure 0.05 MPa;

[0035] c) When the dissolved oxygen (DO) drops below 30%, adjust the aeration rate and stirring speed to control the dissolved oxygen level at 30% until fermentation ends;

[0036] d) The fermentation medium consisted of 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.

[0037] 2.3, 5 L fermenters ybiI T257A Validation of strain production performance

[0038] The L-threonine fermentation yields of different strains were determined and are shown in Table 1. The results indicate that... ybiI After glutamine at position 86 of the gene's amino acid sequence was replaced with leucine, the production of L-threonine was significantly increased compared to the original strain. The strain THRS-YbiI Q86L was then named strain FFTHR-36.

[0039] Table 1: Performance of strains in L-threonine production in a 5 L fermenter

[0040]

[0041] Example 3: Construction of recombinant strain using THRS-6 and fermentation production of L-threonine

[0042] Following the method described in Example 1, strain THRS-6-△ was constructed based on THRS-6. ybiI The original strain and the modified strain, THRS-6-YbiIQ86L, were used. The yields of the original and modified strains were verified by fermentation in a 5 L fermenter, following the fermentation method described in Example 2. The strain THRS-6-YbiIQ86L was named strain FFTHR-37. The L-threonine fermentation yields of different strains are shown in Table 2. This indicates that the mutant YbiI protein can increase the L-threonine fermentation yield in different strains.

[0043] Table 2: Performance of strains in L-threonine production in a 5 L fermenter

[0044]

[0045] The sequence involved in this invention is as follows:

[0046] YbiI protein sequence:

[0047] MASGWANDDAVNEQINSTIEDAIARARGEIPRGESLDECEECGAPIPQARREAIPGVRLCIHCQQEKDLQKPAYTGYNRRGSKDSQLR (SEQ ID NO:1)

[0048] Mutant YbiI protein sequence:

[0049] MASGWANDDAVNEQINSTIEDAIARARGEIPRGESLDECEECGAPIPQARREAIPGVRLCIHCQQEKDLQKPAYTGYNRRGSKDSLLR (SEQ ID NO: 2)

[0050] ybiI Nucleotide sequence of the gene:

[0051] ttaacgtaactggctgtctttcgaacctctgcgattatatcctgtataagctggtttttgtaaatctttctcctgctgacaatgaatacataagcgcacgccaggaatggcttcccgacgggcctgcgggatgggggcaccgcactcttcacattcatccaggctttcgccgcgcggaatttcaccccgagcgcgggcaatcgcatcttcaattgtactgttgatctgttcgttgacggcgtcatcgttagcccaaccggatgccat (SEQ ID NO:3).

[0052] Mutant ybiI Nucleotide sequence of the gene:

[0053] ttaacgtaacaggctgtctttcgaacctctgcgattatatcctgtataagctggtttttgtaaatctttctcctgctgacaatgaatacataagcgcacgccaggaatggcttcccgacgggcctgcgggatgggggcaccgcactcttcacattcatccaggctttcgccgcgcggaatttcaccccgagcgcgggcaatcgcatcttcaattgtactgttgatctgttcgttgacggcgtcatcgttagcccaaccggatgccat (SEQ ID NO:4)

[0054] Pkan-F:

[0055] 5'-AAATACAAATTATACTCGCTATGCATCTCGCCAGTTGTAGAGGCTGGAGCTGCTTC-3' (SEQIDNO:5)

[0056] Pkan-R:

[0057] 5'-CTACACTTAACTGTACAAGTATTGATATGGGGAGGTCGATTCCGGGGATCCGTCGACC-3' (SEQIDNO:6)

[0058] PybiI-F':

[0059] 5'-TTAACGTAACTGGCTGTCTTCGAACCTC-3' (SEQ ID NO: 7)

[0060] PybiI-R':

[0061] 5'-ATGGCATCCGGTTGGGCTAAC-3' (SEQ ID NO: 8)

[0062] PpCP20-F:

[0063] 5'-ATGTCTGAATTAGTTGTTTTCAAAGCAAATGAAC-3' (SEQ ID NO: 9)

[0064] PpCP20-R:

[0065] 5'-GATCCTTCCGTATTTAGCCAGTATGTTCT-3' (SEQ ID NO: 10)

[0066] PybiI-F:

[0067] 5'-AAATACAAATTATACTCGCTATGCATCTCGCCAGTTGTAGTTAACGTAACAGGCTGTCTTTCGAACCTC-3' (SEQ ID NO: 11)

[0068] PybiI-R:

[0069] 5'-CTACACTTAACTGTACAAGTATTGATATGGGGAGGTCGATATGGCATCCGGTTGGGCTAAC-3' (SEQ ID NO: 12)

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

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

2.

2. A mutant ybiI gene, characterized in that, The mutant ybiI gene encodes the mutant YbiI protein as described in claim 1.

3. The application of the mutant YbiI protein of claim 1, or the mutant ybiI gene of claim 2, in increasing threonine production in strains that produce threonine, characterized in that... The strain that produces threonine is Escherichia coli.

4. A recombinant strain that produces high levels of L-threonine, characterized in that, The recombinant strain expresses the mutant YbiI protein of claim 1, or the ybiI gene of the recombinant strain is replaced with the mutant ybiI gene of claim 2, wherein the recombinant strain is Escherichia coli.

5. The recombinant strain with high L-threonine production as described in claim 4, characterized in that, The ybiI gene in the recombinant strain is replaced with the mutant ybiI gene through mutagenesis, site-directed mutagenesis by PCR, or homologous recombination.

6. The method for preparing the recombinant strain with high L-threonine production according to claim 4 or 5, characterized in that, The process includes the following steps: using the mutant ybiI gene as a template, design homologous arm primers PybiI-F / PybiI-R, construct PCR amplification products, transform the PCR amplification products into threonine-producing strains, and obtain recombinant strains by replacing the target region through homologous recombination.

7. A method for producing L-threonine, characterized in that, The process includes the following steps: inoculating the recombinant strain of high L-threonine production as described in claim 4 or 5 onto a seed culture medium 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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