Application of GTP binding protein in preparation of L-valine

Abstract

The invention discloses application of GTP binding protein in preparation of L-valine, and belongs to the technical field of genetic engineering. The application comprises the application of the GTP binding protein or a substance for regulating and controlling the activity or content of the GTP binding protein in preparation of L-valine and / or improvement of the yield of the L-valine, and the amino acid sequence of the protein is SEQ ID NO: 1. According to the present invention, by overexpressing the yihA gene in the CGMCC No.22721 strain, the accumulation concentration of the L-valine in the strain is promoted to be increased from 27.48 g / L to 30.21 g / L, and the yield of the L-valine is increased by 9.95%.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of GTP binding protein in the preparation of L-valine. Background Art

[0002] Since Gourp Besanty first discovered L-valine in pancreatic extract in 1895, the production method of L-valine has undergone countless changes and improvements. So far, there are three mature production methods, namely extraction, chemical synthesis and microbial fermentation. These three production methods have different characteristics and can meet different production needs.

[0003] Breeding efficient strains with the ability to produce L-valine is the key to the industrial application of microbial fermentation. There are currently two main methods for breeding L-amino acid production strains: 1) Rational metabolic engineering: This method mainly uses efficient gene editing technology to systematically transform the L-valine synthesis network in the chassis microorganisms to maximize the redirection of carbon metabolic flux to the L-valine synthesis pathway, mainly including blocking the L-valine degradation pathway, removing the feedback inhibition regulation mechanism of key enzymes in the synthesis pathway, enhancing the supply of precursors, optimizing the balance of chassis cell coenzyme supply, modifying the transmembrane transport system, etc. 2) Irrational adaptive evolution: Adaptive evolution refers to a breeding strategy that changes the growth environment of microorganisms under laboratory conditions, allowing microorganisms to spontaneously mutate in the process of environmental adaptation and complete the evolution process, and then rationally analyzes the evolutionary results. Since this strategy obtains mutant strains in an irrational way and then analyzes them in a rational way, it is a semi-rational breeding strategy that combines rationality and irrationality. Compared with mutation breeding, spontaneous mutations in the adaptive evolution process are screened and strengthened generation by generation due to the limitation of specific culture conditions, so it is more directional and greatly reduces the number of irrelevant mutation samples.

[0004] Systems biology integrates information at different levels to study the interactions between various parts. In recent years, various omics tools have developed rapidly. Among them, transcriptomics is an important discipline that studies the transcription and regulation of the entire gene in cells from the RNA level, which can objectively and truly reflect the key transcription links from gene to protein expression. The combination of systems biology and transcriptomics can better understand the characteristics and response mechanisms of cells under genetic and environmental disturbances. In order to survive in harsh environments for a long time, the expression levels of key driver genes in specific biological processes related to stress are often changed to adapt to the environment. Therefore, studying the differential expression of genes in adapting to environmental stress at the transcriptional level can better understand the mechanism of tolerance of organisms.

[0005] The yihA gene encodes a GTP-binding protein required for normal cell division, and its expression level has increased significantly during the anaerobic adaptive evolution. However, the effect of the enhanced expression intensity of the yihA gene on L-valine production has not been studied. In particular, there is no report on the production of L-valine using Enterobacteriaceae bacteria that overexpress the yihA gene. Summary of the invention

[0006] The technical problem to be solved by the present invention is the application of GTP binding protein in the production of L-valine. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention provides an application, which includes the application of a GTP binding protein or a substance for regulating the activity or content of the GTP binding protein in preparing L-valine and / or increasing the yield of L-valine.

[0009] In the above application, the regulation includes upregulating, enhancing or increasing the expression of the GTP-binding protein encoding gene or upregulating, enhancing or increasing the activity and / or content of the protein.

[0010] In the above application, the substance may be a biological material, and the biological material includes any of the following:

[0011] B1), a nucleic acid molecule encoding the aforementioned GTP-binding protein;

[0012] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0013] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0014] B4), a recombinant cell containing the nucleic acid molecule described in B1), or a recombinant cell containing the expression cassette described in B2), or a recombinant cell containing the recombinant vector described in B3).

[0015] The aforementioned biological materials also fall within the protection scope of the present invention.

[0016] The present invention also provides a recombinant bacterium for producing L-valine, wherein the activity of the GTP binding protein in the recombinant bacterium is enhanced.

[0017] The recombinant bacteria can be prepared by the following method: introducing a substance that upregulates, enhances or improves the expression of the GTP-binding protein encoding gene or upregulates, enhances or improves the activity and / or content of the protein into the starting strain to obtain a recombinant bacteria, wherein the activity of the GTP-binding protein in the recombinant bacteria is higher than that in the starting strain.

[0018] Furthermore, the L-valine production of the recombinant bacteria is higher than that of the starting strain.

[0019] In the above-mentioned recombinant bacteria, the starting strain includes any one of Escherichia coli, Corynebacterium glutamicum, and Pantoea.

[0020] Specifically, the Escherichia coli includes the strain with the registration number of CGMCC No. 22721 and / or Escherichia coli MG1655.

[0021] Specifically, the recombinant bacteria include a recombinant Escherichia coli obtained by inserting the DNA fragment sequence shown in nucleotides 303 to 1060 in SEQ ID NO: 3 into the spacer region of the CGMCC No. 22721 genome that does not encode any gene, while keeping other nucleotide sequences unchanged.

[0022] Specifically, the recombinant bacterium is a recombinant Escherichia coli obtained by inserting the DNA fragment sequence represented by nucleotides 303 to 1060 in SEQ ID NO: 3 into the spacer region of the MG1655 genome that does not encode any gene, while keeping other nucleotide sequences unchanged.

[0023] Specifically, the recombinant bacteria can be constructed according to a method comprising the following steps: introducing the DNA fragment represented by nucleotides 303 to 1060 in SEQ ID NO: 3 into Escherichia coli to obtain a recombinant Escherichia coli. The Escherichia coli includes a strain with a registration number of CGMCC No. 22721 or a wild-type Escherichia coli MG1655. The Escherichia coli is a strain with a registration number of CGMCC No. 22721 or a wild-type Escherichia coli MG1655.

[0024] Specifically, the DNA fragment is introduced into the Escherichia coli by introducing a DNA molecule having a nucleotide sequence of SEQ ID NO:3.

[0025] In the above-mentioned application or nucleic acid molecule or recombinant bacterium, the GTP-binding protein comprises any of the following:

[0026] C1) contains a protein represented by the amino acid sequence of SEQ ID NO: 1;

[0027] C2) a protein having more than 95% identity with the protein shown in C1) and having the same function as the protein shown in C1) obtained by substitution and / or deletion and / or addition of amino acid residues;

[0028] C3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of C1) or C2).

[0029] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0030] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0031] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0032] In the above-mentioned biological material, the expression cassette described in B2) refers to a single-stranded or double-stranded nucleic acid molecule that can express the above-mentioned protein in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule of all regulatory sequences necessary for expressing the nucleic acid molecule of any of the above-mentioned proteins or the DNA of the RNA molecule. The regulatory sequence can guide the coding sequence to express any of the above-mentioned proteins or the DNA of the RNA molecule in a suitable host cell under its compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence and a transcription terminator. At a minimum, the regulatory sequence should include a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule, a regulatory sequence with a joint can be provided. The regulatory sequence can be a suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the protein or the DNA of the RNA molecule. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence can be operably connected to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an mRNA non-translated region that is very important for the translation of the host cell. The leader sequence can be operably connected to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence connected to the amino terminus of the protein, which can guide the DNA encoding the protein or the RNA molecule into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein or the DNA of the RNA molecule into the secretory pathway of the host cell used can be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein or the RNA molecule according to the growth of the host cell. Examples of regulatory sequences are systems that can turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those that allow gene amplification.

[0033] The present invention also provides a whole-cell catalyst, which comprises the aforementioned recombinant bacteria or the aforementioned biological material.

[0034] The present invention also provides a method for producing L-valine, comprising fermenting and producing L-valine using the aforementioned recombinant bacteria or the aforementioned biological material or the aforementioned whole cell catalyst.

[0035] A method for constructing an engineered bacterium for producing L-valine comprises the following steps: enhancing the content and / or activity of a GTP-binding protein in a receptor bacterium.

[0036] In the above method, enhancing the content and / or activity of GTP-binding protein in the recipient bacteria comprises the following steps: introducing a substance that enhances the expression of the GTP-binding protein encoding gene or enhances the activity of the protein or increases the activity content of the protein into the starting strain, so that the expression level of L-valine in the recombinant bacteria is higher than the expression level of L-valine in the starting strain.

[0037] In the above method, the recipient bacteria include Escherichia coli, Corynebacterium glutamicum, and Pantoea.

[0038] Methods for increasing the expression intensity of the yihA gene include but are not limited to using an autonomously replicating plasmid (e.g., pET28a, pTr c99a, pSTV28, etc.) to increase the copy number of the yihA gene; or increasing the copy number of the yihA gene in the chromosome of the chassis microorganism; or using a strong promoter (e.g., Ptrc, BBa-J23100, T7, etc.) and ribosome binding site modification to increase the expression level of the yihA gene; or a combination of the above methods.

[0039] In the above method, the overexpression of the yihA gene means that the expression intensity of the gene is significantly enhanced after modification compared with the original expression intensity of the target gene.

[0040] Specifically, the present invention uses CRISPR / Cas9-mediated gene editing technology to integrate the yihA gene into the spacer region of the Escherichia coli genome that does not encode any gene, and is controlled by the promoter Ptrc to achieve the purpose of enhancing the expression intensity of the yihA gene.

[0041] The present invention utilizes Escherichia coli expressing yihA gene to produce L-valine by fermentation, and provides a method for producing L-valine. The yihA gene is overexpressed in the genome of the strain to enhance the expression intensity of the yihA gene.

[0042] The yihA gene encodes a GTP-binding protein required for normal cell division. The present invention will overexpress the yihA gene in Escherichia coli to enhance the expression intensity of the yihA gene, resulting in a more cost-effective L-valine fermentation production. Specifically, using the wild strain MG1655 as the starting strain, enhancing the expression intensity of the yihA gene causes the L-valine concentration in the strain to increase from 0.001 g / L to 0.056 g / L, and the L-valine yield is significantly improved; using an L-valine engineered strain as the starting strain, enhancing the expression intensity of the yihA gene causes the L-valine accumulation concentration in the strain to increase from 27.48 g / L to 30.21 g / L, and the L-valine yield is increased by 9.95%.

[0043] Storage Instructions:

[0044] Bacteria species: Escherichia coli

[0045] Latin name: Escherichia coli

[0046] Strain ID: YP045

[0047] Classification name: Escherichia coli

[0048] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0049] Abbreviation of depository institution: CGMCC

[0050] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0051] Date of deposit: June 15, 2021

[0052] The registration number of the Collection Center is: CGMCC No.22721. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the pREDCas9 plasmid map.

[0054] Figure 2 This is the plasmid map of pGRB. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0057] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a very significant difference.

[0058] The Escherichia coli MG1655 in the following examples was derived from Beijing Beina Chuanglian Biotechnology Research Institute (BNCC), and the strain number was: BNCC363342.

[0059] The engineered strain CGMCC No.22721 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration.

[0060] Example 1. Construction of genetically engineered bacteria YPVAL-yihA and MG1655-yihA

[0061] 1. Preparation of DNA fragments for homologous recombination

[0062] The recombinant fragment used for overexpression of the yihA gene (the genome sequence is shown in NCBI GeneID: 948358) consists of the upstream and downstream homology arms of the yihA gene (upstream homology arm-downstream homology arm). The primer design software primer5 was used to design the upstream and downstream homology arm primers with the upstream and downstream sequences of the yihA gene as templates, as follows:

[0063] yihA-up-F: 5'-GTAAACAGGCGCTGTTTGTG-3';

[0064] yihA-up-R: 5'-GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAActccccaagggggcgagggg-3';

[0065] yihA-F: 5'-GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCatgagtgcgcctgatattcg-3';

[0066] yihA-R: 5'-ACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGttattcgccgtcctgcgtttc-3';

[0067] yihA-down-F: 5'-CCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATagggttagggtgagggggcg-3';

[0068] yihA-down-R: 5'-GAGCGTACCTCGACATCGTG-3'.

[0069] Using the genomic DNA of the engineering strain CGMCC No.22721 as a template, the upstream homology arm of the yihA gene was amplified with yihA-up-R and yihA-up-F, and the amplification system is shown in Table 1, and 302 bp of the upstream homology arm (i.e., positions 1 to 302 of SEQ ID NO: 3) was obtained. Using the genomic DNA of the engineering strain CGMCC No.22721 as a template, the downstream homology arm of the yihA gene was amplified with yihA-down-R and yihA-down-F, and the amplification system is shown in Table 1, and 297 bp of the downstream homology arm (i.e., positions 1061 to 1357 of SEQ ID NO: 3) was obtained. Using the genomic DNA of the engineering strain CGMCC No.22721 as a template, the CDS region and promoter P of the yihA gene were amplified with yihA-R and yihA-F. trc and rrnB terminator (i.e., positions 303 to 1060 of SEQ ID NO: 3), named P trc -yihA-T rrnB The amplification system is shown in Table 1.

[0070] Table 1. PCR amplification system

[0071] Components Volume (50 μL) DNA template 1μL Upstream primer (10 μmol / L) 1μL Downstream primer (10 μmol / L) 1μL dNTP mixture (10mmol / L) 4μL 5×Buffer 10μL HS enzyme (5U / μL) 0.5μL ddH2O 32.5μL

[0072] The upstream primer yihA-down-F of the upstream homology arm and the downstream primer yihA-down-R of the downstream homology arm were used as amplification primers, and the upstream homology arm and the downstream homology arm were used as P trc -yihA-T rrnBOverlap PCR was performed for the amplification template to prepare the recombinant fragment. Overlap PCR reaction conditions (Bao Bio PrimeSTAR HS enzyme): pre-denaturation (95°C) for 5 min; then 30 cycles: denaturation (98°C) for 10 s, annealing ((Tm-3 / 5)°C) for 15 s, extension at 72°C (this enzyme activity extends about 1 kb per 1 min); continue extension at 72°C for 10 min; maintain (4°C). The overlapping PCR reaction system is shown in Table 2, and the DNA fragment used for homologous recombination is obtained. The DNA fragment used for homologous recombination P trc The nucleotide sequence of -yihA-Trrnb is SEQ ID NO: 3, wherein positions 1 to 302 are the upstream homology arm of the yihA gene (302 bp), positions 303 to 376 are P trc promoter (74 bp), positions 377 to 973 are the yihA gene (597 bp), positions 974 to 1060 are the rrnB terminator region (87 bp), and positions 1061 to 1357 are the downstream homologous arms of the yihA gene (297 bp).

[0073] Table 2. Overlapping PCR reaction system

[0074] Components Volume (50 μL) template 2μL Upstream primer of upstream homology arm (10 μmol / L) 1μL Downstream homology arm downstream primer (10μmol / L) 1μL dNTP mixture (10mmol / L) 4μL 5×Buffer 10μL HS enzyme (5U / μL) 0.5μL ddH2O 31.5μL

[0075] Note: The template consists of the amplified fragments of the upstream and downstream homology arms and the target gene (P trc -yihA-T rrnB ) are equimolar and the total amount does not exceed 10ng.

[0076] 2. Construction of pGRB-sgRNA plasmid

[0077] pGRB was purchased from Addgene, catalog number #71539, with pUC18 as the backbone, including promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100 mg / L), and cultured at 37°C.

[0078] The pGRB plasmid was constructed by recombining a DNA fragment containing the target sequence with a linearized vector fragment.

[0079] (1) Target sequence and primer design

[0080] CRISPR RGEN Tools was used to design the target sequence (PAM: 5'-NGG-3'), and the primers for amplifying the sgRNA fragment for the aforementioned target sequence (forward primer F structure: 5'-linearized vector end sequence (34 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector end sequence (34 bp)-3', reverse primer R structure: a primer that is reverse complementary to the forward primer R), as follows:

[0081] gRNA-yihA-F: 5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTgcccccttggggagagggttGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3';

[0082] gRNA-yihA-R: 5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACaaccctctccccaagggggcACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3';

[0083] In the above primers, lowercase letters represent target sequences.

[0084] (2) Preparation of DNA fragments containing target sequences

[0085] Prepare DNA fragments containing the target sequence by annealing single-stranded DNA. Reaction conditions: pre-denaturation at 95°C for 5 min; annealing at 30-50°C for 1 min. Annealing reaction system: gRNA-yihA-F (10 μmol / L) 10 μL, gRNA-yihA-R (10 μmol / L) 10 μL.

[0086] (3) Preparation of linear vector

[0087] The linearization of the vector was carried out by the inverse PCR amplification method. The amplification primers are as follows: pGRB-F: 5'-ACTAGTATTATACCTAGGACTGAGC-3'; pGRB-R: 5'-GTTTTAGAGCTAGAAATAGCAAGTT-3'. The PCR reaction system is shown in Table 1. PCR reaction program (Bao Bio PrimeSTAR HS enzyme): pre-denaturation (95℃) for 5min; then 30 cycles: denaturation (98℃) for 10s, annealing ((Tm-3 / 5)℃) for 15s, extension at 72℃ (this enzyme activity extends about 1kb for 1min); continue extension at 72℃ for 10min; maintain (4℃).

[0088] (4) Recombination reaction

[0089] The recombinant system is shown in the following table. All recombinant enzymes used are ClonExpress IIOne Step Cloning Kit series enzymes, recombination conditions: 37°C, 30 min.

[0090] Table 3. Recombination system

[0091]

[0092] (5) Plasmid transformation

[0093] Take 10 μL of the reaction solution in "(4) Recombination Reaction" and add it to 100 mL of DH5α competent cells. Mix gently and place on ice for 20 min. Heat shock at 42°C for 45-90 s. Place on ice for 2-3 min. Add 900 μL of SOC and resuscitate at 37°C for 1 h. Centrifuge at 8000 rpm for 2 min. Discard part of the supernatant and keep about 200 μL to resuspend the cells. Apply the cells to a plate containing 100 mg / L ampicillin. Invert the plate and culture at 37°C overnight. After a single colony grows on the plate, identify the positive recombinants by colony PCR.

[0094] (6) Clone identification

[0095] The PCR-positive colonies were inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight to maintain the bacteria. The plasmids were extracted and identified by enzyme digestion.

[0096] The plasmid identified as correctly inserted was named pGRB-sgRNA. The pGRB-sgRNA can transcribe the gRNA with the nucleotide sequence of SEQ ID NO: 4, thereby forming a complex with the Cas9 protein, and recognize the target gene target site through base pairing and PAM to achieve the target DNA double-strand break.

[0097] 3. Transformation of plasmids and recombinant DNA fragments

[0098] pREDCas9 was purchased from Addgene, catalog number #71541, carrying the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage and the Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg / L), and cultured at 32°C.

[0099] (1) Transformation with pREDCas9

[0100] The pREDCas9 plasmid was electroporated into the electroporation competent state of the starting strain by electroporation. After the bacteria were revived and cultured, they were spread on LB plates containing spectinomycin and cultured overnight at 32°C. Single colonies grown on the resistant plates were subjected to colony PCR using identification primers to screen positive recombinants.

[0101] (2) Preparation of electroporation competent cells of target strain containing pREDCas9

[0102] The positive recombinants in "(1) pREDCas9 transformation" were cultured at 32°C until OD 600nm =0.1-0.2, add 0.1M IPTG (to make the final concentration 0.1mM), and continue to culture until OD600nm =0.6-0.7, and the competent state was prepared. The purpose of adding IPTG is to induce the expression of the recombinase on the pREDCas9 plasmid. The culture medium and preparation process required for competent state preparation refer to conventional standard operations to obtain electroporated competent cells containing pREDCas9.

[0103] (3) Transformation of pGRB and recombinant DNA fragments

[0104] The pGRB-sgRNA and donor DNA fragment were simultaneously electrotransformed into the electrocompetent cells containing pREDCas9 obtained in "(2) Preparation of electrocompetent cells of target strain containing pREDCas9". The revived bacteria after electrotransformation were spread on LB plates containing ampicillin and spectinomycin and cultured at 32°C overnight. Colony PCR verification was performed using identification primers yihA-up-F / yihA-R1 and yihA-F2 / yihA-down-R, and positive recombinants were screened and maintained.

[0105] yihA-up-F: 5'-GTAAACAGGCGCTGTTTGTG-3';

[0106] yihA-R1: 5'-ACCAGGTATCCAGTTTCTGC-3';

[0107] yihA-F2: 5'-TTGAAGTGGCTTTTGCAGGC-3';

[0108] yihA-down-R: 5'-GAGCGTACCTCGACATCGTG-3'.

[0109] (4) Elimination of plasmids

[0110] ① Elimination of pGRB

[0111] The positive recombinants obtained in "(3) Transformation of pGRB and recombinant DNA fragments" were cultured overnight in LB medium containing 0.2% arabinose, and then appropriately diluted and spread on LB plates containing spectinomycin resistance, and cultured overnight at 32°C. Single colonies were picked and streaked one by one onto LB plates containing ampicillin and spectinomycin resistance, and single colonies that did not grow on the ampicillin plate but grew on the spectinomycin resistance plate were selected to obtain positive recombinants.

[0112] ② Elimination of pREDCas9 plasmid

[0113] The positive recombinants obtained in "① Elimination of pGRB" were transferred to LB liquid medium without resistance, cultured at 42°C overnight, and spread on LB plates without resistance after appropriate dilution, and cultured at 37°C overnight. Single colonies were picked and streaked one by one onto LB plates containing spectinomycin resistance and no resistance, and single colonies that did not grow on spectinomycin resistance plates but grew on non-resistance plates were selected to obtain positive single colonies.

[0114] The positive colonies obtained above were sent for sequencing, and the strain with the correct sequencing results was named recombinant bacteria YPVAL-yihA. Recombinant bacteria YPVAL-yihA is a recombinant Escherichia coli obtained by inserting the DNA fragment sequence shown in nucleotides 303 to 1060 in SEQ ID NO:3 into the spacer region of the CGMCC No. 22721 genome that does not encode any gene, while keeping other nucleotide sequences unchanged.

[0115] According to the above method, the wild-type strain MG1655 was used as the starting strain to obtain the recombinant bacterium MG1655-yihA. The recombinant bacterium MG1655-yihA is a recombinant Escherichia coli obtained by inserting the DNA fragment sequence represented by nucleotides 303 to 1060 in SEQ ID NO: 3 into the spacer region of the MG1655 genome that does not encode any gene, while keeping other nucleotide sequences unchanged.

[0116] Example 2. Differences in L-valine production performance between MG1655-yihA and YPVAL-yihA modified by overexpressing the yihA gene

[0117] The composition of the slant medium is: glucose 1-5g / L, peptone 5-10g / L, beef extract 5-10g / L, yeast powder 1-5g / L, NaCl 1-2.5g / L, agar 15-20g / L, and the rest is water, pH 7.0-7.2;

[0118] The composition of the seed culture medium is: yeast extract 4-6g / L, peptone 8-10g / L, NaCl 8-10g / L, and the rest is water, pH 7.0-7.2.

[0119] The fermentation medium composition is: yeast extract 1-3g / L, K 2 HPO 4 2-5g / L, MgSO 4 7H 2 0 0.2-0.5g / L, anhydrous citric acid 0.5-1g / L, FeSO 4 7H 2 O 15-20mg / L, MnSO 4 7H 2 O 5-10 mg / L, V H0.01-0.05mg / L, V B1 、V B3 、V B5 、V B12 1-3 mg / L each, the rest is water, pH 7.0-7.2.

[0120] The experiment was repeated three times, and the steps for each repetition were as follows:

[0121] 1. Slant culture: YPVAL-yihA, MG1655-yihA, starting strain CGMCC No. 22721 and starting strain MG1655 stored at -80°C were streaked onto activated slant surfaces, cultured at 37°C for 12 h, and subcultured once;

[0122] 2. Shake flask seed culture: Use an inoculation loop to scrape a ring of slant seeds and inoculate it into a 500mL Erlenmeyer flask containing 30mL of seed culture medium, seal it with nine layers of gauze, and culture it at 37℃ and 200rpm for 7-10h;

[0123] 3. Shake flask fermentation: inoculate 10-15% of the volume of the seed culture solution into a 500mL Erlenmeyer flask (final volume of 30mL) filled with fermentation medium, seal with nine layers of gauze, and culture at 37°C, 200r / min with shaking. During the fermentation process, maintain the pH at 7.0-7.2 by adding ammonia water; add 60% (m / v) glucose solution to maintain the fermentation; the fermentation period is 26-30h.

[0124] 4. Take the fermentation supernatant after 26-30 hours of fermentation in "3. Shake flask fermentation culture" and determine the L-valine concentration in the fermentation supernatant by HPLC.

[0125] Table 4. Shake flask fermentation results of YPVAL-yihA and MG1655-yihA strains

[0126] strain <![CDATA[OD 600nm ]]> Valine(g / L) Difference Analysis CGMCC 22721 20.36±0.57 27.48±0.56 YPVAL-yihA 17.73±0.24 30.21±0.25 P＜0.01 MG1655 35.46±0.25 0.012±0.01 MG1655-yihA 34.43±0.56 0.056±0.02 P＜0.01

[0127] The results are shown in Table 4. The enhancement of yihA gene expression intensity has a certain effect on the growth of wild strains and engineered strains. At the same time, the concentration of L-valine in MG1655-yihA increased from 0.012 g / L to 0.056 g / L, and the yield of L-valine increased significantly. The concentration of L-valine in YPVAL-yihA increased from 27.48 g / L to 30.21 g / L, and the yield of L-valine increased by 9.93%. The results show that enhancing the expression intensity of yihA gene in Escherichia coli can significantly improve the L-valine production performance of engineered strains.

[0128] SEQ ID NO: 1 (yihA encoded amino acid)

[0129] MSAPDIRHLPSDTGIEVAFAGRSNAGKSSALNTLTNQKSLARTSKTPGRTQLINLFEVADGKRLVDLPGYGYAEVPEEMKRKWQRALGEYLEKRQSLQGLVVLMDIRHPLKDLDQQMIEWAVDSNIAVLVLLTKADKLASGARKAQLNMVREAVLAFNGDVQVETFSSLKKQGVDKLRQKLDTWFSEMQPVEETQDGE*。

[0130] SEQ ID NO: 2 (Coding region of yihA gene)

[0131] 5’-ATGAGTGCGCCTGATATTCGCCACCTACCTTCCGATACCGGAATTGAAGTGGCTTTTGCAGGCCGTTCCAACGCAGGTAAATCCAGCGCGCTGAACACGCTGACTAACCAGAAAAGCCTGGCTCGTACCTCAAAAACCCCAGGGCGCACCCAGCTTATCAACCTGTTTGAAGTGGCTGACGGCAAGCGTCTGGTTGACTTGCCTGGGTACGGTTATGCGGAAGTCCCGGAAGAGATGAAGCGCAAATGGCAGCGTGCGCTCGGCGAATACCTCGAAAAACGTCAGAGCCTGCAAGGTCTGGTGGTGCTAATGGATATTCGCCATCCGCTGAAAGATTTGGATCAGCAGATGATTGAGTGGGCGGTAGACAGCAATATCGCCGTTCTGGTGCTGCTGACCAAAGCGGACAAACTGGCAAGCGGCGCACGTAAAGCGCAATTGAATATGGTGCGTGAAGCTGTACTGGCGTTTAACGGTGATGTGCAGGTTGAAACGTTTTCTTCGTTGAAGAAACAAGGCGTGGACAAGCTGCGGCAGAAACTGGATACCTGGTTTAGCGAGATGCAGCCTGTAGAAGAAACGCAGGACGGCGAATAA-3'。

[0132] SEQ ID NO: 3 (P trc -yihA-T rrnB Gene integration fragment)

[0133]

[0134] SEQ ID NO:4 (pGRB-sgRNA sequence)

[0135] 5′-GCCCCCUUGGGGAGAGGGUU-3′.

[0136] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application, characterized in that, The application includes the application of the GTP binding protein or a substance for regulating the activity or content of the GTP binding protein in preparing L-valine and / or increasing the yield of L-valine.

2. The use according to claim 1, characterized in that: The regulation includes regulating, enhancing or increasing the expression of the GTP-binding protein encoding gene or regulating, enhancing or increasing the activity and / or content of the protein.

3. Biomaterial, characterized in that The biological material includes any of the following: B1), a nucleic acid molecule encoding the GTP-binding protein according to any one of claims 1-2; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant cell containing the nucleic acid molecule described in B1), or a recombinant cell containing the expression cassette described in B2), or a recombinant cell containing the recombinant vector described in B3).

4. A recombinant bacterium for producing L-valine, characterized in that: The activity of the GTP-binding protein in the recombinant bacteria is enhanced.

5. The recombinant bacterium according to any one of claim 4, characterized in that The recombinant bacteria include any one of Escherichia coli, Corynebacterium glutamicum and Pantoea.

6. The use according to any one of claims 1-2 or the nucleic acid molecule according to claim 3 or the recombinant bacterium according to any one of claims 4-5, characterized in that: The GTP binding protein comprises any of the following: C1) contains a protein represented by the amino acid sequence of SEQ ID NO: 1; C2) a protein having more than 95% identity with the protein shown in C1) and having the same function as the protein shown in C1) obtained by substitution and / or deletion and / or addition of amino acid residues; C3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in any one of C1) or C2).

7. A whole cell catalyst, characterized in that The whole-cell catalyst comprises the recombinant bacteria described in any one of claims 4-5 or the biological material described in claim 3.

8. A method for producing L-valine, comprising fermenting and producing L-valine using the recombinant bacteria according to any one of claims 4 to 5, the biological material according to claim 3, or the whole cell catalyst according to claim 7.

9. A method for constructing an engineered bacterium for producing L-valine, comprising the following steps: enhancing the content and / or activity of a GTP-binding protein in a receptor bacterium.

10. The method according to claim 9, wherein the recipient bacteria comprises Escherichia coli, Corynebacterium glutamicum, and Pantoea.

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