Promoter mutants and their applications

Abstract

The present application provides a promoter, an expression cassette comprising the promoter, an expression vector comprising the promoter or the expression cassette, a microorganism comprising the promoter, the expression cassette or the expression vector, and their uses in regulating the expression intensity of a target gene, biocatalysis, or biofermentation. The present application also provides a method for increasing amino acid production.

Description

Technical Field

[0001] The present application relates to the fields of genetic engineering and fermentation. Specifically, the present application provides promoter mutants and applications thereof. Background Art

[0002] L-Leucine is an essential branched-chain amino acid critical to maintaining human health. It plays a vital role in multiple industries: in the medical field, it is used to prepare amino acid infusions to stimulate insulin production, maintain nutritional balance in critically ill patients, treat various diseases, and synthesize anti-cancer drugs. In the food industry, it serves as an additive, enhancing the taste and nutritional value of food while also aiding in muscle repair after exercise. In the beauty industry, L-Leucine acts as a natural moisturizing ingredient, promoting healthy skin and hair. In agriculture, as part of amino acid fertilizers, it promotes plant growth and improves soil quality.

[0003] With the surge in market demand for L-leucine, improving its production process has become particularly critical. Currently, the main methods for producing L-leucine include extraction, chemical synthesis, enzymatic catalysis, and microbial fermentation. Microbial fermentation is considered to be more suitable for large-scale industrial applications due to its environmental friendliness, low cost, and easy-to-control reaction process. However, the complex metabolic pathways and low catalytic efficiency and low specificity of key enzymes in the synthesis pathway during microbial synthesis of L-leucine have limited its development at the industrial level.

[0004] Therefore, improving the catalytic efficiency of key enzymes in the L-leucine synthesis pathway is an effective means to increase the level of L-leucine synthesis by microorganisms. Summary of the Invention

[0005] In a first aspect, the present application provides a promoter comprising a nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 1.

[0006] In a second aspect, the present application provides an expression cassette comprising the promoter described in the first aspect.

[0007] In a third aspect, the present application provides an expression vector comprising the promoter described in the first aspect or the expression cassette described in the second aspect.

[0008] In a fourth aspect, the present application provides a microorganism comprising the promoter described in the first aspect, the expression cassette described in the second aspect, or the expression vector described in the third aspect.

[0009] In a fifth aspect, the present application provides the use of the promoter described in the first aspect, the expression cassette described in the second aspect, the expression vector described in the third aspect, or the microorganism described in the fourth aspect in regulating the expression intensity of a target gene, biocatalysis, or biofermentation.

[0010] In one or more embodiments of the present application, regulating the expression intensity of the target gene is to enhance the expression intensity of the target gene.

[0011] In one or more embodiments of the present application, the target gene is a protein-encoding gene.

[0012] In one or more embodiments of the present application, the biocatalysis is the catalysis of a substrate by an enzyme, and the expression of the enzyme is regulated by the promoter described in the first aspect.

[0013] In one or more embodiments of the present application, the biological fermentation is fermentation of amino acids.

[0014] In a sixth aspect, the present application provides a method for increasing amino acid production, which comprises using the microorganism described in the fourth aspect. DETAILED DESCRIPTION

[0015] α-Isopropylmalate synthase (IPMS), encoded by the leuA gene, is the core rate-limiting enzyme in the L-leucine biosynthetic pathway. Existing technical means are to enhance the efficient expression of IPMS by increasing the copy number, but this can cause homologous sequences to cause genomic instability problems through mechanisms such as recombination, rearrangement or mutation. The inventors of the present application constructed a promoter library by randomly mutating the promoter of the leuA gene (e.g., the constitutive promoter M1-93), and then screened for promoter mutants that can enhance the expression of the leuA gene, and constructed a recombinant strain containing the promoter to increase the yield and conversion rate of L-leucine. The inventors of the present application optimized the promoter strength to increase the expression level of IPMS without increasing the gene copy number, thereby avoiding the potential genetic risks caused by the increase in gene copy number as much as possible. In addition, the promoter mutants obtained by screening can also enhance the expression of other genes (e.g., leuB, leuCD, leuDH or tyrB genes), and / or increase the yield and conversion rate of other amino acids (e.g., L-valine).

[0016] The practice of this application will employ, unless otherwise indicated, conventional techniques of genetic engineering, microbiology, biochemistry, and analytical chemistry within the skill of the art.

[0017] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0018] Unless otherwise specified, all experimental reagents were commercially available products.

[0019] definition

[0020] As used herein, the term "promoter" refers to a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for RNA polymerase specific binding and transcription initiation. Most of them are located upstream of the transcription start point of the structural gene. The promoter itself is not transcribed.

[0021] As used herein, the term "constitutive promoter" refers to a promoter that can activate gene expression in all tissues, whose regulation is not affected by external conditions, and the expression of the activated gene is continuous but does not show temporal and spatial specificity.

[0022] As used herein, the term "promoter strength" refers to the efficiency and rate of promoter-driven transcription. In gene expression regulation, the level of promoter strength determines the transcription level of the gene in the cell.

[0023] As used herein, the term "identity" refers to the percentage of identical residues in nucleotide or amino acid sequence variants after sequence alignment and introduction of a gap. Methods and computer programs for comparison are well known in the art. "At least 90% identity" as described herein refers to any value of 90% to 100% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a numerical value or range between any of the above values. For purposes of the present invention, "identity" can be calculated by comparing two compared sequences in a comparison window. The comparison of sequences makes it possible to determine the number of shared positions (such as nucleotides) of two sequences in the comparison window. Then, the number of shared positions is divided by the total number of positions in the comparison window and multiplied by 100 to obtain percent identity. The determination of sequence identity percentage can be completed manually or using a known computer program.

[0024] In a first aspect, the present application provides a promoter comprising a nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 1.

[0025] In one or more embodiments of the present application, the promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0026] In one or more embodiments of the present application, the promoter consists of the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 1.

[0027] In one or more embodiments of the present application, the promoter consists of a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0028] In one or more embodiments of the present application, any nucleotide sequence having the above-mentioned identity in which a portion of the sequence in the promoter sequence is deleted, modified, substituted or inserted should be understood to be included in the scope of the present application, as long as the sequence has the same or similar transcriptional activity as the promoter. For example, a polynucleotide having a meaningless sequence added to or at the end of the nucleotide sequence of SEQ ID NO: 1, or having a portion of the sequence deleted from or at the end of the nucleotide sequence of SEQ ID NO: 1 is also included in the scope of the present application, as long as it has the same or similar transcriptional activity as the promoter.

[0029] In one or more embodiments of the present application, the promoter consists of the nucleotide sequence shown in SEQ ID NO: 1.

[0030] In one or more embodiments of the present application, the promoter is a promoter obtained by random mutation of the constitutive promoter M1-93, and is a mutant of the M1-93 promoter.

[0031] In a second aspect, the present application provides an expression cassette comprising the promoter described in the first aspect.

[0032] In one or more embodiments of the present application, the expression cassette further comprises a target gene. The promoter of the present application can be operably linked to the target gene.

[0033] In one or more embodiments of the present application, the target gene can be any nucleic acid sequence, preferably a gene encoding a protein with a specific function.

[0034] In a third aspect, the present application provides an expression vector comprising the promoter described in the first aspect or the expression cassette described in the second aspect.

[0035] In one or more embodiments of the present application, the expression vector can be a recombinant expression vector, which comprises the promoter described in the first aspect and optionally comprises a multiple cloning site or at least one restriction enzyme site downstream of the promoter. When it is necessary to express a target gene, the target gene is connected to a suitable multiple cloning site or restriction enzyme site, thereby operably connecting the target gene to the promoter. Methods for preparing recombinant vectors are well known to those skilled in the art. Methods well known to those skilled in the art can be used to construct expression vectors containing the promoter described in the present application and / or the target gene.

[0036] In a fourth aspect, the present application provides a microorganism comprising the promoter described in the first aspect, the expression cassette described in the second aspect, or the expression vector described in the third aspect.

[0037] In one or more embodiments of the present application, the microorganism is a bacterium or a fungus.

[0038] In one or more embodiments of the present application, the microorganism is Escherichia coli, Klebsiella pneumoniae, Corynebacterium glutamicum, Bacillus subtilis or yeast.

[0039] In one or more embodiments of the present application, the promoter and / or target gene are transduced into the microorganism. For example, based on CRISPR / Cas9 gene editing, the promoter and / or target gene are transduced into the genome of the microorganism.

[0040] In a fifth aspect, the present application provides the use of the promoter described in the first aspect, the expression cassette described in the second aspect, the expression vector described in the third aspect, or the microorganism described in the fourth aspect in regulating the expression intensity of a target gene, biocatalysis, or biofermentation.

[0041] In one or more embodiments of the present application, regulating the expression intensity of the target gene is to enhance the expression intensity of the target gene.

[0042] In one or more embodiments of the present application, the target gene is a protein-encoding gene.

[0043] In one or more embodiments of the present application, the protein is a related enzyme in an amino acid metabolic pathway, such as a related enzyme in an amino acid synthesis and / or transport pathway.

[0044] In one or more embodiments of the present application, the protein is a related enzyme in the branched-chain amino acid metabolic pathway, such as a related enzyme in the branched-chain amino acid synthesis and / or transport pathway.

[0045] In one or more embodiments of the present application, the related enzyme is α-isopropylmalate synthase, β-isopropylmalate dehydrogenase, α-isopropylmalate isomerase, leucine dehydrogenase, acetohydroxy acid synthase, hydroxy acid reductoisomerase, dihydroxy acid dehydratase, branched-chain amino acid aminotransferase, branched-chain amino acid efflux protein, L-leucine transporter, pyridine nucleotide transhydrogenase, ubiquitin kinase, glutamate dehydrogenase, phosphoketolase, leucine export protein or aromatic amino acid aminotransferase.

[0046] In one or more embodiments of the present application, the target gene is leuA, leuB, leuCD, leuDH, bcd, ilvBN, ilvC, ilvD, ilvE, brnFE, yeaS, pntAB, coaA, rocG, fxpk, leuE or tyrB gene.

[0047] Among them, the leuA gene encodes α-isopropylmalate synthase.

[0048] Among them, the leuB gene encodes β-isopropylmalate dehydrogenase.

[0049] Among them, the leuCD gene encodes α-isopropylmalate isomerase.

[0050] Among them, the leuDH gene encodes leucine dehydrogenase.

[0051] Among them, the bcd gene encodes leucine dehydrogenase, such as the bcd gene of Bacillus subtilis.

[0052] Among them, the ilvBN gene encodes acetohydroxyacid synthase.

[0053] Among them, the ilvC gene encodes hydroxyacid reductoisomerase.

[0054] Among them, the ilvD gene encodes dihydroxyacid dehydratase.

[0055] Among them, the ilvE gene encodes branched-chain amino acid aminotransferase.

[0056] Among them, the brnFE gene encodes a branched-chain amino acid efflux protein.

[0057] Among them, the yeaS gene encodes the L-leucine transporter.

[0058] Among them, the pntAB gene encodes pyridine nucleotide transhydrogenase.

[0059] Among them, the coaA gene encodes ubiquitin kinase.

[0060] Among them, the rocG gene encodes glutamate dehydrogenase.

[0061] Among them, the fxpk gene encodes phosphoketolase.

[0062] Among them, the leuE gene encodes a leucine exporter.

[0063] Among them, the tyrB gene encodes aromatic amino acid aminotransferase.

[0064] In one or more specific embodiments of the present application, the target gene is leuA, leuB, leuCD, leuDH or tyrB gene.

[0065] In one or more embodiments of the present application, the biocatalysis is the catalysis of a substrate by an enzyme, and the expression of the enzyme is regulated by the promoter described in the first aspect, for example, the catalysis of α-ketoisovalerate by α-isopropylmalate synthase.

[0066] In one or more embodiments of the present application, the biocatalysis may take different forms, including but not limited to crude enzyme catalysis, pure enzyme catalysis, and whole-cell conversion.

[0067] In one or more embodiments of the present application, the crude enzyme may be an unpurified enzyme preparation obtained by culturing (eg, fermentation culture or induction culture) the microorganism described in the fourth aspect, crushing or lysing the cells, and then centrifuging the supernatant.

[0068] In one or more embodiments of the present application, the pure enzyme is a pure enzyme preparation obtained by treating the crude enzyme through various purification techniques (eg, centrifugation, filtration, chromatography, etc.).

[0069] In one or more embodiments of the present application, the whole-cell transformation method utilizes intact cells for biotransformation. For example, the intact cells are the microorganisms described in the fourth aspect.

[0070] In one or more embodiments of the present application, the biological fermentation is fermentation of amino acids.

[0071] In one or more embodiments of the present application, the biological fermentation is fermentation of branched-chain amino acids.

[0072] In one or more embodiments of the present application, the promoter described in the first aspect of the present application increases the expression of the leuA gene.

[0073] In one or more embodiments of the present application, the promoter described in the first aspect of the present application increases the expression of the leuB gene.

[0074] In one or more embodiments of the present application, the promoter described in the first aspect of the present application increases the expression of the leuCD gene.

[0075] In one or more embodiments of the present application, the promoter described in the first aspect of the present application increases the expression of the leuDH gene.

[0076] In one or more embodiments of the present application, the promoter described in the first aspect of the present application increases the expression of the tyrB gene.

[0077] In one or more embodiments of the present application, the promoter described in the first aspect of the present application increases the expression of α-isopropylmalate synthase (IPMS), thereby increasing the yield and conversion rate of L-leucine.

[0078] In one or more embodiments of the present application, the promoter described in the first aspect of the present application increases the yield and conversion rate of L-valine.

[0079] In one or more embodiments of the present application, the promoter described in the first aspect of the present application can be used to enhance the expression of multiple target genes.

[0080] In one or more embodiments of the present application, the use of the promoter described in the first aspect of the present application avoids the risk caused by an increase in gene copy number.

[0081] In one or more embodiments of the present application, the use of the promoter described in the first aspect of the present application maintains the genetic stability of the strain.

[0082] In one or more specific embodiments of the present application, the present application uses Ls003 bacteria as the chassis strain, and first uses error-prone PCR technology to perform random mutations on the parent promoter (e.g., M1-93 promoter); secondly, a repair template containing a promoter mutant (e.g., ycjv::pM1-93*-leuA*(R308S T407I G462D)) is constructed; finally, a strain containing a promoter mutant is constructed, and a fermentation test is performed to screen promoter mutant strains with improved production and conversion rate of amino acids (e.g., L-leucine).

[0083] In a sixth aspect, the present application provides a method for increasing amino acid production, which comprises using the microorganism described in the fourth aspect.

[0084] Those skilled in the art can use the microorganisms described in the fourth aspect to produce amino acids through conventional technical means in the art.

[0085] In one or more embodiments of the present application, the microorganism may be cultured to produce the amino acid.

[0086] In one or more embodiments of the present application, the gene encoding the amino acid can be introduced into the microorganism, and then the microorganism containing the gene encoding the amino acid is cultured to produce the amino acid, wherein the gene encoding the amino acid is regulated by the promoter described in the first aspect.

[0087] In one or more embodiments of the present application, the amino acid is a branched-chain amino acid.

[0088] In one or more embodiments of the present application, the amino acid is leucine, valine, isoleucine, alanine or arginine.

[0089] In one or more embodiments of the present application, the amino acid is an L-amino acid or a D-amino acid.

[0090] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application and is not intended to limit the present invention in any respect. Those skilled in the art can make various modifications and variations to the embodiments described. Example

[0091] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0092] 1. The strains and plasmids constructed in this application are detailed in Table 1, and the primers used are detailed in Table 2.

[0093] Table 1. Strains and plasmids used in the present invention

[0094]

[0095] Table 2. Primers used in the present invention

[0096]

[0097] 2. Sources of relevant biological materials

[0098] 2 × Phanta Mix high-fidelity enzyme is a product of Novezan, product number: P525-01.

[0099] QuickMutation TM The gene random mutagenesis kit was a product of Beyotime, with the number D0219S.

[0100] Example 1. Obtaining an M1-93 promoter mutation library

[0101] The Ls003 strain is a strain obtained by knocking out the ilvE, leuDH and avtA genes based on the strain SvalM029 disclosed in Chinese patent application No. 202210710799.8.

[0102] The Ls004 strain was modified from Ls003 by knocking out the ycjv gene, replacing the leuA gene with the leuA*(R308S T407I G462D) gene, and replacing the leuA promoter with pM1-93. leuA*(R308S T407I G462D) is a mutant of the wild-type leuA, whose amino acid sequence is NCBI Reference Sequence: WP_000082850.1. pM1-93 represents the M1-93 promoter.

[0103] Using Biyuntian's QuickMutation TM Gene random mutagenesis kit was used, using primers pM1-93-F / pM1-93-R and Ls004 genome as template to PCR amplify the fragment containing the pM1-93 mutant.

[0104] The PCR reaction system (50 μL) consisted of: 30 μL of ddH2O, 2 μL of upstream primer (10 mM), 2 μL of downstream primer (10 mM), 1 μL of DNA template, 5 μL of RandomMut buffer (10×), 4 μL of Mutation Enhancer (10×), 5 μL of dNTPs (2.5 mM each), and 1 μL of RandomMut DNA polymerase;

[0105] The PCR reaction procedure was as follows: Step 1: 94°C, 3 min; Step 2: 94°C, 30 s, 55°C, 30 s; Step 3: 72°C, 1.5 min; this step was repeated 30 times, followed by 72°C, 10 min; and Step 4: Storage at 4°C. The pM1-93 mutant library (containing the pM1-93* mutant fragment) was amplified by PCR.

[0106] Example 2. Obtaining a mutant library of α-isopropylmalate synthase pM1-93

[0107] (1) Construction of leuA editing elements for genomic integration of the pM1-93 library: Using specific primers ΔycjV-up-F / ΔycjV-up-R, the Ls004 genome was used as a template to amplify the upstream homology arm UP fragment (ΔycjV-UP) by PCR technology; using specific primers leuA-F / ΔycjV-down-R, the Ls004 genome was used as a template to amplify the leuA*-DOWN fragment containing leuA* (R308S T407I G462D) and the downstream homology arm DOWN fragment by PCR; using overlapping PCR technology to connect ΔycjV-UP, the pM1-93* mutant fragment, and leuA*-DOWN to obtain the homologous recombinant fragment UP-pM1-93*-leuA*-DOWN (ΔycjV::pM1-93*-leuA-donor).

[0108] The PCR amplification system and reaction procedures used for amplifying the UP fragment and the leuA*-DOWN fragment are as follows:

[0109] The PCR reaction system (50 μL) consisted of: 20 μL of ddH O, 2 μL of upstream primer (10 mM), 2 μL of downstream primer (10 mM), 1 μL of plasmid template, and 25 μL of 2 × Phanta Mix high-fidelity enzyme;

[0110] The PCR reaction procedure was as follows: step 1: 95°C for 3 minutes; step 2: 95°C for 15 seconds, 60°C for 10 seconds; step 3: 72°C for 3 minutes; this step was repeated 30 times, followed by 72°C for 5 minutes; and step 4: storage at 4°C. PCR amplification yielded ΔycjV-UP and leuA*-DOWN.

[0111] (2) The overlapping PCR reaction system (50 μL) consisted of: 25 μL of 2 × Phanta Mix high-fidelity enzyme, 1 μL each of ΔycjV-UP, pM1-93* mutant fragment, and leuA*-DOWN fragment, 2 μL each of ΔycjV-up-F (10 mM) and ΔycjV-down-R (10 mM), and 18 μL of ddH2O;

[0112] The overlapping PCR reaction procedure was as follows: step 1: 95°C for 3 min; step 2: 95°C for 15 s, 60°C for 10 s; step 3: 72°C for 2 min; this step was repeated 30 times, followed by 72°C for 5 min; and step 4: storage at 4°C. The resulting ΔycjV::pM1-93*-leuA*-donor fragment was obtained.

[0113] Example 3. Obtaining a recombinant strain containing a pM1-93* mutant and an α-isopropylmalate synthase mutant

[0114] (1) Preparation of Ls003 competent cells: Pick a single clone of the Ls003 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL LB liquid medium. Cultivate at 37°C and 200 rpm until the OD 550 The precipitate was resuspended in 20 mL of 10% glycerol and centrifuged at 5000 r / min at 4°C for 6 min. The above steps were repeated twice, and finally 1 mL of 10% glycerol was added to resuspend the cells. Aliquots were made into 100 μL tubes and stored at -80°C for later use.

[0115] (2) Transformation of pECas9 plasmid: 1 μL of pECas9 plasmid and 100 μL of Ls003 competent cells were added to an electroporation cup, mixed well, and placed on ice for 5 min; the electroporation cup was wiped dry, electroporated at 2.5 KV, and 1 mL of pre-cooled LB medium was immediately added, pipetted several times, mixed well, and transferred to a test tube; cultured at 37°C, 200 r / min for 1-2 h; 100 μL of bacterial solution was evenly spread on a solid LB plate with ampicillin (final concentration of 100 μg / mL) and cultured at 37°C overnight.

[0116] (3) Preparation of competent cells of Ls003 / pECas9 strain: Pick a single clone of Ls003 / pECas9 strain and inoculate it into 100 mL of LB medium containing 5% L-arabinose (100 μg / mL Amp resistance), and culture at 37°C, 200 rpm for 3 h until OD 600 = 0.5; ice bath for 20 min; transfer to a 50 mL sterile centrifuge tube, centrifuge at 5000 rpm at 4°C for 6 min; discard the supernatant, resuspend the pellet in 20 mL of 10% glycerol, and centrifuge at 5000 rpm at 4°C for 6 min; repeat the above step twice, and finally add 1 mL of 10% glycerol to resuspend the bacteria to obtain Ls003 / pECas9 competent cells, aliquot 100 μL / tube, and store at -80°C for later use.

[0117] Preparation of pTarget-ΔycjV-sgRNA plasmid: Using the pTarget plasmid as a template, amplify the pTarget-ΔycjV-sgRNA fragment using the ΔycjV-sgRNA-F / ΔycjV-sgRNA-R primers. Transform the fragment into DH5α competent cells and sequence the fragment. Extract the plasmid from the correctly sequenced single clone to obtain the pTarget-ΔycjV-sgRNA plasmid. The PCR reaction system and reaction procedure were the same as step (1) of Example 2.

[0118] (4) Transformation of pTarget-ΔycjV-sgRNA plasmid and ΔycjV::pM1-93*-leuA*-donor fragment: Take 2 μL of the above-constructed ΔycjV::pM1-93*-leuA*-donor fragment and pTarget-ΔycjV-sgRNA plasmid, and add them to the electroporation cup with 100 μL Ls003 / pECas9 competent cells, mix well and place on ice for 5 min; culture at 37℃, 200 r / min for 1-2 h; centrifuge and spread all on solid LB plates with ampicillin and spectinomycin (final concentrations of 100 μg / mL and 100 μg / mL, respectively), and culture at 37℃ overnight. The single colony grown on the plate is the strain containing the α-isopropylmalate synthase pM1-93* mutant. All the single colonies grown on the plate were preserved and stored in a -80℃ refrigerator for later use in 48-deep-well plate testing.

[0119] Example 4. 48-deep-well plate fermentation of mutants

[0120] The recombinant strains containing the α-isopropylmalate synthase mutant and the pM1-93* mutant, along with a control strain, Ls004, were inoculated into 48-deep-well plates containing leucine fermentation medium. The cultures were cultured at 37°C and 220 rpm for 48 hours, after which the fermentation broth was removed and the L-leucine content was determined using HPLC. The recombinant strains containing the α-isopropylmalate synthase mutant and the pM1-93* mutant differed from the control strain, Ls004, only in the promoter regulating the leuA* (R308S T407I G462D) gene (see Table 1). The results showed that compared to the control strain, Ls004, the strain containing the pM1-93* mutant of α-isopropylmalate synthase showed significantly increased leucine accumulation. The pM1-93* mutant was sequenced in this strain, and the nucleotide sequence of the pM1-93* mutant is shown in SEQ ID NO: 1.

[0121] HPLC detection method for L-leucine: After the sample is diluted 10 times, centrifuge it at 12000 r / min for 10 min, filter the supernatant with a filter membrane and then analyze it by HPLC.

[0122] Chromatographic column: Primesep 100 column

[0123] The mobile phase for determination was prepared as follows: 0.05% sulfuric acid, 35% acetonitrile, and 64.95% water;

[0124] Detector: UV detector

[0125] Column temperature: 35°C

[0126] Detection wavelength: 200nm

[0127] Fermentation medium: 20 g / L glucose, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 0.2 g / L betaine, 0.2 g / L valine, 0.2 g / L isoleucine, 1 mL of 1× trace element stock solution, and 15 mL of biotin stock solution (0.4 g / L).

[0128] Table 3. 1× trace element mother solution formula

[0129]

[0130] Example 5. Shake flask fermentation of leuA gene containing pM1-93* mutant

[0131] The recombinant strain containing the α-isopropylmalate synthase mutant and the pM1-93* mutant and the control strain Ls004 were inoculated into LB liquid test tubes and cultured at 37°C, 200 rpm overnight. The above culture solution was transferred to 50 mL of fermentation medium at a 1% inoculum volume. After culturing at 37°C, 200 rpm for 48 h, 1 mL of fermentation broth was collected and the L-leucine production was detected using the HPLC method described in Example 4.

[0132] Table 4. L-leucine production

[0133]

[0134] Fermentation results showed that the leucine fermentation yield of the recombinant strains containing the pM1-93* mutant was improved, among which the mutant strain Ls007 had the best effect (the promoter mutant was named pM1-93-3), with a shake flask L-leucine yield of 2.35 g / L and a conversion rate of 18%. Compared with the promoter before mutation, the yield increased by 82% and the conversion rate increased by 80%.

[0135] Example 6. Application of the pM1-93 mutant in the expression of other genes for L-leucine production

[0136] 6.1 Obtaining the Ls011-Ls012 recombinant strain

[0137] Construction of leuB and leuCD editing elements for genomic integration:

[0138] (1) PCR amplification yielded ΔyciQ-UP(pM1-93 / pM1-93-3), leuBCD(pM1-93 / pM1-93-3), and ΔyciQ-DOWN:

[0139] Using the Ls004 genome as a template, the specific primers ΔyciQ-up-F / ΔyciQ-up-R (pM1-93) were used to amplify the upstream homology arm UP fragment containing the M1-93 promoter by PCR technology, namely ΔyciQ-UP (pM1-93); using the Ls004 genome as a template, the specific primers ΔyciQ-up-F / ΔyciQ-up-R (pM1-93-3) were used to amplify the upstream homology arm UP fragment containing the pM1-93-3 promoter by PCR technology, namely ΔyciQ-UP (pM1-93-3); using the Ls004 genome as a template, the specific primers leuBCD-F (pM1-93) / leuBCD-R, the leuBCD fragment containing pM1-93 was amplified by PCR technology, namely leuBCD(pM1-93); using the Ls004 genome as a template, specific primers leuBCD-F(pM1-93-3) / leuBCD-R were used to amplify the leuBCD fragment containing pM1-93-3 by PCR technology, namely leuBCD(pM1-93-3); using the specific primers ΔyciQ-down-F / ΔyciQ-down-R, the downstream homology arm DOWN fragment, namely ΔyciQ-DOWN, was amplified by PCR using the Ls004 genome as a template.

[0140] (2) Overlap PCR amplification of homologous recombination fragments UP-pM1-93-leuBCD-DOWN (ΔyciQ::pM1-93-leuBCD-donor) and UP-pM1-93-3-leuBCD-DOWN (ΔyciQ::pM1-93-3-leuBCD-donor):

[0141] ① Using overlapping PCR technology, ΔyciQ-UP(pM1-93), leuBCD(pM1-93), and ΔyciQ-DOWN were connected to obtain the homologous recombinant fragment UP-pM1-93-leuBCD-DOWN (ΔyciQ::pM1-93-leuBCD-donor);

[0142] The overlapping PCR reaction system (50 μL) consisted of: 25 μL of 2 × Phanta Mix high-fidelity enzyme, 1 μL each of ΔyciQ-UP (pM1-93), leuBCD (pM1-93), and ΔyciQ-DOWN fragments, 2 μL each of ΔyciQ-up-F (10 mM) and ΔyciQ-down-R (10 mM), and 18 μL of ddH2O;

[0143] The overlapping PCR reaction procedure was as follows: step 1: 95°C for 3 min; step 2: 95°C for 15 s, 60°C for 10 s; step 3: 72°C for 2 min; this step was repeated 30 times, followed by 72°C for 5 min; and step 4: storage at 4°C. The resulting ΔyciQ::pM1-93-leuBCD-donor / ΔyciQ::pM1-93-3-leuBCD-donor fragment was obtained.

[0144] ② Use overlapping PCR technology to connect ΔyciQ-UP (pM1-93-3), leuBCD (pM1-93-3) and ΔyciQ-DOWN to obtain the homologous recombinant fragment UP-pM1-93-3-leuBCD-DOWN (ΔyciQ::pM1-93-3-leuBCD-donor).

[0145] The overlapping PCR reaction system (50 μL) consisted of: 25 μL of 2 × Phanta Mix high-fidelity enzyme, 1 μL each of ΔyciQ-UP (pM1-93-3), leuBCD (pM1-93-3), and ΔyciQ-DOWN fragments, 2 μL each of ΔyciQ-up-F (10 mM) and ΔyciQ-down-R (10 mM), and 18 μL of ddH2O;

[0146] The overlapping PCR reaction procedure was as follows: step 1: 95°C for 3 min; step 2: 95°C for 15 s, 60°C for 10 s; step 3: 72°C for 2 min; this step was repeated 30 times, followed by 72°C for 5 min; and step 4: storage at 4°C. The resulting ΔyciQ::pM1-93-leuBCD-donor / ΔyciQ::pM1-93-3-leuBCD-donor fragment was obtained.

[0147] (3) PCR amplification of the pTarget-ΔyciQ-sgRNA fragment: Using pTarget as a template, the pTarget-ΔyciQ-sgRNA-F / ΔyciQ-sgRNA-R primers were used to amplify the pTarget-ΔyciQ-sgRNA fragment.

[0148] The PCR reaction system (50 μL) consisted of: 20 μL of ddH2O, 2 μL of upstream primer (10 mM), 2 μL of downstream primer (10 mM), 1 μL of plasmid template, and 25 μL of 2 × Phanta Mix high-fidelity enzyme;

[0149] The PCR reaction program was as follows: step 1: 95°C, 3 min; step 2: 95°C, 15 s, 60°C, 10 s; step 3: 72°C, 3 min; this step was repeated 30 cycles, 72°C, 5 min; and step 4: storage at 4°C.

[0150] (4) Transformation: Fragment the pTarget-ΔyciQ-sgRNA and transform it into DH5α competent cells to obtain the pTarget-ΔyciQ-sgRNA targeting plasmid.

[0151] (5) Obtaining recombinant strains containing leuA mutants:

[0152] The pTarget-ΔyciQ-sgRNA plasmid and ΔyciQ::pM1-93-leuBCD-donor fragment constructed above were electroporated into Ls007 for gene editing. A single colony that was sequenced correctly was designated Ls011 (Ls007 ycjv::pM1-93-leuBCD) and subsequently tested in shake flasks.

[0153] The pTarget-ΔyciQ-sgRNA plasmid and ΔyciQ::pM1-93-3-leuBCD-donor fragment constructed above were electroporated into Ls007 for gene editing. A single colony that was sequenced correctly was designated Ls012 (Ls007 ycjv::pM1-93-3-leuBCD) and subsequently tested in shake flasks.

[0154] 6.2 Shake flask fermentation of recombinant strains Ls011 and Ls012

[0155] The above strains Ls011 and Ls012 and the control strain Ls007 were respectively inoculated into LB liquid test tubes and cultured at 37°C, 200 r / min overnight. The above culture fluids were transferred to 50 mL of fermentation medium at a 1% inoculum volume. After culturing at 37°C, 200 r / min for 48 h, 1 mL of fermentation fluid was taken and the L-leucine production was detected using the HPLC method described in Example 4.

[0156] Table 5. L-leucine production of strains Ls011 and Ls012

[0157]

[0158] In this example, the leuB and leuCD genes were overexpressed based on the Ls007 strain, using the original pM1-93 promoter and the pM1-93-3 mutant promoter, respectively. Fermentation results showed that overexpression of the leuB and leuCD genes increased L-leucine production, with the mutant strain Ls012 achieving the best results, with a shake flask L-leucine production of 3.77 g / L and a conversion rate of 24.2%. Compared with the Ls011 strain containing the original pM1-93, the yield increased by 10% and the conversion rate increased by 9.5%.

[0159] Example 7. Application of pM1-93 mutant in L-valine production

[0160] The M1-93 promoter of the LeuDH gene in the Sval026 strain disclosed in Chinese Patent Application No. CN202010401422.5 was replaced with pM1-93-3, and the strain was named Sval026*. Strain Sval026* was fermented and cultured according to Example 13 of Chinese Patent Application No. CN202010401422.5.

[0161] The results showed that the control strain Sval026 could produce 1.8 g / L of L-valine under anaerobic conditions for 4 days, with a sugar-acid conversion rate of 0.56 mol / mol; the promoter mutant strain Sval026* could produce 2.1 g / L of L-valine under anaerobic conditions for 4 days, with a sugar-acid conversion rate of 0.61 mol / mol. Compared with the Sval026 strain containing the original pM1-93, the yield increased by 16.7% and the conversion rate increased by 8.9%.

[0162] In summary, the M1-93-3 promoter mutant of the present invention has the effect of enhancing the expression of various bio-based products, including but not limited to genes encoding related enzymes in the amino acid metabolic pathway.

[0163] It is understood that although the inventions described herein are described in the aforementioned specific forms, these inventions are not limited to the specific contents described in these specific forms. It is obvious to those skilled in the art that various equivalent variations may be made to the technical features of the inventions described herein without departing from the spirit of the inventions described herein, and such variations should fall within the scope of the inventions.

Claims

1. A promoter consisting of the nucleotide sequence shown in SEQ ID NO:

1.

2. An expression cassette comprising the promoter according to claim 1.

3. An expression vector comprising the promoter according to claim 1 or the expression cassette according to claim 2.

4. A microorganism comprising the promoter according to claim 1, the expression cassette according to claim 2 or the expression vector according to claim 3. The microorganism according to claim 4 , wherein the microorganism is a bacterium or a fungus.

6. The microorganism according to claim 4, wherein the microorganism is Escherichia coli, Klebsiella pneumoniae, Corynebacterium glutamicum, Bacillus subtilis or yeast.

7. Use of the promoter according to claim 1, the expression cassette according to claim 2, the expression vector according to claim 3, or the microorganism according to any one of claims 4 to 6 in regulating the expression intensity of a target gene, biocatalysis, or biofermentation; Wherein, regulating the expression intensity of the target gene is to enhance the expression intensity of the target gene, and the target gene is leuA, leuB, leuCD or leuDH gene; The biocatalysis is the catalysis of a substrate by an enzyme, the expression of which is regulated by the promoter according to claim 1, wherein the enzyme is α-isopropylmalate synthase, β-isopropylmalate dehydrogenase, α-isopropylmalate isomerase or leucine dehydrogenase; The biological fermentation is fermentation of amino acids, and the amino acid is L-leucine or L-valine.

8. The method according to claim 7, wherein the target gene is a gene encoding a protein, and the protein is α-isopropylmalate synthase, β-isopropylmalate dehydrogenase, α-isopropylmalate isomerase or leucine dehydrogenase.

9. A method for increasing amino acid production, comprising using the microorganism according to any one of claims 4 to 6; wherein the amino acid is L-leucine or L-valine.

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