Promoter mutant and application thereof

Abstract

The invention provides a promoter, an expression cassette containing the promoter, an expression vector containing the promoter or the expression cassette, a microorganism containing the promoter, the expression cassette or the expression vector, and application of the promoter, the expression cassette or the expression vector in regulation and control of target gene expression intensity, biological catalysis or biological fermentation. The invention further provides a method for increasing the yield of amino acid.

Description

Technical Field

[0001] The present application relates to the field 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 that is critical to maintaining human health. It plays an important role in many 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 a variety of diseases, and synthesize anti-cancer drugs; in the food industry, it is used as an additive to not only enhance the taste of food and improve nutritional value, but also help repair muscles after exercise; in the beauty field, L-Leucine, as a natural moisturizing ingredient, promotes the health of skin and hair; in the agricultural field, as part of amino acid fertilizers, it can promote plant growth and soil improvement.

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

[0004] Therefore, improving the catalytic efficiency of key enzymes in the L-leucine biosynthesis 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 as 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, the 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 biosynthetic pathway of L-leucine. The existing technical means is to enhance the efficient expression of IPMS by enhancing the copy number, but this will cause the homologous sequence to cause genomic instability through mechanisms such as recombination, rearrangement or mutation. The inventors of the present application construct a promoter library by randomly mutating the promoter of the leuA gene (such as the constitutive promoter M1-93), and then screen the promoter mutants that can enhance the expression of the leuA gene, and construct a recombinant strain containing the promoter to increase the yield and conversion rate of L-leucine. The inventors of the present application optimize the promoter strength, without increasing the gene copy number, to increase the expression level of IPMS, thereby avoiding the potential genetic risks caused by the increase in the gene copy number as much as possible. In addition, the promoter mutants obtained by screening can also enhance the expression of other genes (such as leuB, leuCD, leuDH or tyrB genes), and / or increase the yield and conversion rate of other amino acids (such as L-valine).

[0016] The practice of the present 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. definition

[0019] 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.

[0020] As used herein, the term "constitutive promoter" refers to a promoter that can initiate gene expression in all tissues, whose regulation is not affected by external conditions, and the expression of the initiated gene is continuous but does not show spatiotemporal specificity.

[0021] As used herein, the term "promoter strength" refers to the efficiency and rate of promoter-driven transcription. In gene expression regulation, the strength of the promoter determines the transcription level of the gene in the cell.

[0022] 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 spaces. 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 aligned sequences in a comparison window. The alignment of the sequence enables the determination of the number of common positions (such as nucleotides) of the two sequences in the comparison window. Then, the number of common positions is divided by the total number of positions in the comparison window and multiplied by 100 to obtain the identity percentage. The determination of the sequence identity percentage can be completed manually or using a known computer program.

[0023] In a first aspect, the present application provides a promoter comprising a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 1.

[0024] 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.

[0025] 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.

[0026] In one or more embodiments of the present application, the promoter consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:1.

[0027] In one or more embodiments of the present application, any nucleotide sequence with the above identity in which a part 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 inside or at the end of the nucleotide sequence of SEQ ID NO: 1, or having a part of the sequence deleted inside 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.

[0028] In one or more embodiments of the present application, the promoter consists of the nucleotide sequence shown in SEQ ID NO:1.

[0029] 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.

[0030] In a second aspect, the present application provides an expression cassette comprising the promoter described in the first aspect.

[0031] 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.

[0032] In one or more embodiments of the present application, the target gene may be any nucleic acid sequence, preferably a gene encoding a protein with a specific function.

[0033] 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.

[0034] 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 site downstream of the promoter. When it is necessary to express the target gene, the target gene is connected into a suitable multiple cloning site or restriction site, so that the target gene is operably connected to the promoter. The method for preparing the recombinant vector is well known to those skilled in the art. Methods well known to those skilled in the art can be used to construct an expression vector containing the promoter and / or the target gene described in the present application.

[0035] 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.

[0036] In one or more embodiments of the present application, the microorganism is a bacterium or a fungus.

[0037] In one or more embodiments of the present application, the microorganism is Escherichia coli, Klebsiella pneumoniae, Corynebacterium glutamicum, Bacillus subtilis or yeast.

[0038] In one or more embodiments of the present application, the promoter and / or the target gene are transduced into the microorganism. For example, based on CRISPR / Cas9 gene editing, the promoter and / or the target gene are transduced into the genome of the microorganism.

[0039] 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.

[0040] In one or more embodiments of the present application, the regulating the expression intensity of the target gene is to enhance the expression intensity of the target gene.

[0041] In one or more embodiments of the present application, the target gene is a protein encoding gene.

[0042] 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.

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

[0044] In one or more embodiments of the present application, the related enzyme is α-isopropylmalate synthase, β-isopropylmalate dehydrogenase, α-isopropylmalate isomerase, leucine dehydrogenase, acetohydroxyacid synthase, hydroxyacid reductoisomerase, dihydroxyacid 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.

[0045] 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.

[0046] Among them, the leuA gene encodes α-isopropylmalate synthase.

[0047] Among them, the leuB gene encodes β-isopropylmalate dehydrogenase.

[0048] Among them, the leuCD gene encodes α-isopropylmalate isomerase.

[0049] Among them, the leuDH gene encodes leucine dehydrogenase.

[0050] Among them, the bcd gene encodes leucine dehydrogenase, such as the bcd gene of Bacillus subtilis.

[0051] Among them, the ilvBN gene encodes acetohydroxyacid synthase.

[0052] Among them, the ilvC gene encodes hydroxyacid reductoisomerase.

[0053] Among them, the ilvD gene encodes dihydroxyacid dehydratase.

[0054] Among them, the ilvE gene encodes branched-chain amino acid aminotransferase.

[0055] Among them, the brnFE gene encodes a branched-chain amino acid efflux protein.

[0056] Among them, the yeaS gene encodes an L-leucine transporter.

[0057] Among them, the pntAB gene encodes pyridine nucleotide transhydrogenase.

[0058] Among them, the coaA gene encodes ubiquitin kinase.

[0059] Among them, the rocG gene encodes glutamate dehydrogenase.

[0060] Among them, the fxpk gene encodes phosphoketolase.

[0061] Among them, the leuE gene encodes a leucine exporter.

[0062] Among them, the tyrB gene encodes aromatic amino acid aminotransferase.

[0063] In one or more specific embodiments of the present application, the target gene is leuA, leuB, leuCD, leuDH or tyrB gene.

[0064] 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.

[0065] 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.

[0066] 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 centrifuging the supernatant.

[0067] 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.).

[0068] In one or more embodiments of the present application, the "whole cell transformation method" is to use intact cells for biological transformation. For example, the intact cells are the microorganisms described in the fourth aspect.

[0069] In one or more embodiments of the present application, the biological fermentation is fermentation of amino acids.

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

[0071] 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.

[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 leuB 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 leuCD 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 leuDH 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 tyrB 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 α-isopropylmalate synthase (IPMS), thereby increasing the yield and conversion rate of L-leucine.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] In one or more specific embodiments of the present application, the present application adopts Ls003 bacteria as the chassis strain, firstly uses error-prone PCR technology to perform random mutations on the parent promoter (for example, M1-93 promoter); secondly, a repair template containing a promoter mutant (for example, 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 (for example, L-leucine).

[0082] 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.

[0083] Those skilled in the art can use the microorganisms described in the fourth aspect to produce amino acids through conventional techniques in the art.

[0084] In one or more embodiments of the present application, the microorganism may be cultured to produce the amino acid.

[0085] 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.

[0086] In one or more embodiments of the present application, the amino acid is a branched-chain amino acid.

[0087] In one or more embodiments of the present application, the amino acid is leucine, valine, isoleucine, alanine or arginine.

[0088] In one or more embodiments of the present application, the amino acid is an L-amino acid or a D-amino acid.

[0089] It should be understood that the above detailed description is only for enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments. Example

[0090] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0091] 1. The strains and plasmids constructed in this application are detailed in Table 1, and the primers used are detailed in Table 2.

[0092] Table 1. Strains and plasmids used in the present invention

[0093]

[0094] Table 2. Primers used in the present invention

[0095]

[0096] 2. Sources of relevant biological materials

[0097] 2 × Phanta Mix high-fidelity enzyme is a product of Novezan, product number: P525-01.

[0098] QuickMutation TM The gene random mutagenesis kit is a product of Bio-Time, with the serial number D0219S.

[0099] Example 1. Obtaining the M1-93 promoter mutation library

[0100] 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.

[0101] The Ls004 strain was obtained by performing the following transformations on the basis of Ls003: the ycjv gene was knocked out, the leuA coding gene was replaced with the leuA*(R308S T407I G462D) coding gene, and the promoter regulating the leuA coding gene was replaced with pM1-93. Among them, leuA*(R308S T407I G462D) is a mutant of wild-type leuA, and the amino acid sequence accession number of wild-type leuA is: NCBI Reference Sequence: WP_000082850.1; pM1-93 is the M1-93 promoter.

[0102] Use QuickMutation from Biyuntian TM Gene random mutagenesis kit, using pM1-93-F / pM1-93-R primers, Ls004 genome as template, PCR amplification of the fragment containing pM1-93 mutant.

[0103] The PCR reaction system (50 μL) was: 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;

[0104] The PCR reaction program 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 for 30 cycles, 72°C, 10 min; and step 4: stored at 4°C. The pM1-93 mutant library (containing the pM1-93* mutant fragment) was obtained by PCR amplification.

[0105] Example 2. Obtaining a mutant library of α-isopropylmalate synthase pM1-93

[0106] (1) Construction of leuA editing elements for genomic integration of the pM1-93 library: Using specific primers ΔycjV-up-F / ΔycjV-up-R and Ls004 genome as template, the upstream homology arm UP fragment (ΔycjV-UP) was amplified by PCR technology; using specific primers leuA-F / ΔycjV-down-R and Ls004 genome as template, the leuA*-DOWN fragment containing leuA* (R308S T407I G462D) and the downstream homology arm DOWN fragment was amplified by PCR; using overlapping PCR technology, the ΔycjV-UP, the pM1-93* mutant fragment, and the leuA*-DOWN were connected to obtain the homologous recombinant fragment UP-pM1-93*-leuA*-DOWN (ΔycjV::pM1-93*-leuA-donor).

[0107] Among them, the PCR amplification system and reaction procedure used for amplification of the UP fragment and leuA*-DOWN fragment are as follows:

[0108] The PCR reaction system (50 μL) was: 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;

[0109] The PCR reaction procedure 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 for 30 cycles, 72°C, 5 min; and step 4: stored at 4°C. ΔycjV-UP and leuA*-DOWN were obtained by PCR amplification.

[0110] (2) The overlapping PCR reaction system (50 μL) was as follows: 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;

[0111] The overlapping 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, 2 min; this step was repeated for 30 cycles, 72°C, 5 min; and step 4: stored at 4°C. The ΔycjV::pM1-93*-leuA*-donor fragment was obtained.

[0112] Example 3. Obtaining a recombinant strain containing a pM1-93* mutant and an α-isopropylmalate synthase mutant

[0113] (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 pH value was about 0.5-0.6, and the tube was placed in an ice bath for 20 min. The tube was transferred into a 50 mL sterile centrifuge tube and centrifuged at 4°C, 5000 r / min for 6 min. The supernatant was discarded and the precipitate was resuspended in 20 mL 10% glycerol and centrifuged at 4°C, 5000 r / min for 6 min. The above operation was repeated twice, and finally 1 mL 10% glycerol was added to resuspend the cells. The tube was aliquoted into 100 μL tubes and stored at -80°C for later use.

[0114] (2) Transformation of pECas9 plasmid: Take 1 μL of pECas9 plasmid and 100 μL of Ls003 competent cells and add them to the electroporation cup, mix well and place on ice for 5 min; wipe the electroporation cup dry, shock with 2.5 KV and immediately add 1 mL of pre-cooled LB medium, pipette several times, mix well and transfer to a test tube; culture at 37°C, 200 r / min for 1-2 h; take 100 μL of bacterial solution and evenly spread it on a solid LB plate with ampicillin (final concentration of 100 μg / mL) and culture at 37°C overnight.

[0115] (3) Preparation of competent cells of Ls003 / pECas9 strain: Pick a single clone of Ls003 / pECas9 strain and inoculate it into 100 mL 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 4°C, 5000 r / min for 6 min; discard the supernatant, resuspend the precipitate with 20 mL 10% glycerol, centrifuge at 4°C, 5000 r / min for 6 min; repeat the above step twice, and finally add 1 mL 10% glycerol to resuspend the bacteria to obtain Ls003 / pECas9 competent cells, and dispense 100 μL / tube and store at -80°C for later use.

[0116] Preparation of pTarget-ΔycjV-sgRNA plasmid: Using pTarget plasmid as template, ΔycjV-sgRNA-F / ΔycjV-sgRNA-R primers were used to amplify the pTarget-ΔycjV-sgRNA fragment, which was transformed into DH5α competent cells and sequenced for verification. The plasmid extracted from the correctly sequenced single clone was the pTarget-ΔycjV-sgRNA plasmid. The PCR reaction system and reaction procedure were the same as step (1) of Example 2.

[0117] (4) Transformation of pTarget-ΔycjV-sgRNA plasmid and ΔycjV::pM1-93*-leuA*-donor fragment: Take 2 μL of the ΔycjV::pM1-93*-leuA*-donor fragment and pTarget-ΔycjV-sgRNA plasmid constructed above, 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 plate 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 testing in a 48-deep-well plate.

[0118] Example 4. 48-deep-well plate fermentation of mutants

[0119] The above-mentioned recombinant strain containing α-isopropylmalate synthase mutant and pM1-93* mutant and control strain Ls004 were inoculated into a 48-deep well plate containing leucine fermentation medium liquid, and the fermentation liquid was taken out after culturing at 37°C and 220 r / min for 48 hours, and the content of L-leucine was detected by HPLC. Among them, the recombinant strain containing α-isopropylmalate synthase mutant and pM1-93* mutant and the control strain Ls004 only differ in the promoter regulating the leuA*(R308S T407I G462D) gene (see Table 1). The results showed that compared with the control strain Ls004, the leucine accumulation of strain Ls007 containing α-isopropylmalate synthase pM1-93* mutant was significantly increased, and the strain was sequenced for pM1-93* mutant, and the nucleotide sequence of pM1-93* mutant was SEQ ID NO:1.

[0120] 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 measure the sample by HPLC.

[0121] Chromatographic column: Primesep 100 column

[0122] The mobile phase for determination was prepared as follows: 0.05% sulfuric acid, 35% acetonitrile, and 64.95% water;

[0123] Detector: UV detector

[0124] Column temperature: 35°C

[0125] Detection wavelength: 200nm

[0126] 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 biotin stock solution (0.4 g / L).

[0127] Table 3. 1× trace element master solution formula

[0128]

[0129] Example 5. Shake flask fermentation of leuA gene containing pM1-93* mutant

[0130] 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 r / min overnight. The above culture solution was transferred to 50 mL of fermentation medium at a 1% inoculum amount, and after culturing at 37°C, 200 r / min for 48 h, 1 mL of fermentation solution was taken and the L-leucine production was detected using the HPLC method described in Example 4.

[0131] Table 4. L-Leucine production

[0132]

[0133] The 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). The L-leucine production in the shake flask reached 2.35 g / L and the conversion rate reached 18%, which was an 82% increase in the promoter production and an 80% increase in the conversion rate compared to the original promoter.

[0134] Example 6. Application of pM1-93 mutant in the expression of other genes for producing L-leucine

[0135] 6.1 Obtaining the Ls011-Ls012 recombinant strain

[0136] Construction of leuB and leuCD editing elements for genomic integration:

[0137] (1) PCR amplification to obtain ΔyciQ-UP (pM1-93 / pM1-93-3), leuBCD (pM1-93 / pM1-93-3) and ΔyciQ-DOWN:

[0138] 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, i.e., leuBCD(pM1-93), was amplified by PCR technology; 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, i.e., leuBCD(pM1-93-3), by PCR technology; using the specific primers ΔyciQ-down-F / ΔyciQ-down-R, with the Ls004 genome as a template, the downstream homologous arm DOWN fragment, i.e., ΔyciQ-DOWN, was amplified by PCR.

[0139] (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):

[0140] ①Use overlapping PCR technology to connect ΔyciQ-UP(pM1-93), leuBCD(pM1-93) and ΔyciQ-DOWN to obtain the homologous recombinant fragment UP-pM1-93-leuBCD-DOWN (ΔyciQ::pM1-93-leuBCD-donor);

[0141] The overlapping PCR reaction system (50 μL) was: 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;

[0142] The overlapping 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, 2 min; this step was repeated for 30 cycles, 72°C, 5 min; and step 4: stored at 4°C. The ΔyciQ::pM1-93-leuBCD-donor / ΔyciQ::pM1-93-3-leuBCD-donor fragment was obtained.

[0143] 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).

[0144] The overlapping PCR reaction system (50 μL) was: 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;

[0145] The overlapping 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, 2 min; this step was repeated for 30 cycles, 72°C, 5 min; and step 4: stored at 4°C. The ΔyciQ::pM1-93-leuBCD-donor / ΔyciQ::pM1-93-3-leuBCD-donor fragment was obtained.

[0146] (3) PCR amplification of pTarget-ΔyciQ-sgRNA fragment: Using pTarget as a template, ΔyciQ-sgRNA-F / ΔyciQ-sgRNA-R primers were used to amplify the pTarget-ΔyciQ-sgRNA fragment.

[0147] The PCR reaction system (50 μL) was: 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;

[0148] 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 for 30 cycles, 72°C, 5 min; and step 4: stored at 4°C.

[0149] (4) Transformation: Fragment the pTarget-ΔyciQ-sgRNA and transfer it into DH5α competent cells to obtain the pTarget-ΔyciQ-sgRNA targeting plasmid.

[0150] (5) Obtaining recombinant strains containing leuA mutants:

[0151] The pTarget-ΔyciQ-sgRNA plasmid and ΔyciQ::pM1-93-leuBCD-donor fragment constructed above were electroporated into Ls007 for gene editing. The single colony with correct sequencing was named Ls011 (Ls007 ycjv::pM1-93-leuBCD) and was tested in shake flasks later.

[0152] The pTarget-ΔyciQ-sgRNA plasmid and ΔyciQ::pM1-93-3-leuBCD-donor fragment constructed above were electroporated into Ls007 for gene editing. The single colony with correct sequencing was named Ls012 (Ls007 ycjv::pM1-93-3-leuBCD) and was tested in shake flasks later.

[0153] 6.2 Shake flask fermentation of recombinant strains Ls011 and Ls012

[0154] The above strains Ls011 and Ls012 and the control group strain Ls007 were inoculated into LB liquid test tubes, respectively, and cultured at 37°C, 200 r / min overnight. The above culture solutions were transferred to 50 mL of fermentation medium at a 1% inoculum amount, and cultured at 37°C, 200 r / min for 48 h. Then, 1 mL of fermentation solution was taken and the L-leucine production was detected using the HPLC method described in Example 4.

[0155] Table 5. L-leucine production of strains Ls011 and Ls012

[0156]

[0157] In this embodiment, leuB and leuCD genes were overexpressed on the basis of Ls007 strain, and the pM1-93 original promoter and pM1-93-3 mutant promoter were used respectively. The fermentation results showed that the overexpression of leuB and leuCD genes increased the L-leucine production, among which the mutant strain Ls012 had the best effect, with the shake flask L-leucine production reaching 3.77 g / L and the conversion rate reaching 24.2%. Compared with the Ls011 strain containing the original pM1-93, the production was increased by 10% and the conversion rate was increased by 9.5%.

[0158] Example 7. Application of pM1-93 mutant in L-valine production

[0159] 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*. The strain Sval026* was fermented and cultured with reference to Example 13 in Chinese Patent Application No. CN202010401422.5.

[0160] The results showed that the control strain Sval026 could produce 1.8 g / L of L-valine after 4 days of fermentation under anaerobic conditions, and the sugar-acid conversion rate was 0.56 mol / mol; the promoter mutant strain Sval026* could produce 2.1 g / L of L-valine after 4 days of fermentation under anaerobic conditions, and the sugar-acid conversion rate was 0.61 mol / mol. Compared with the Sval026 strain containing the original pM1-93, the yield was increased by 16.7% and the conversion rate was increased by 8.9%.

[0161] In summary, it can be seen that 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.

[0162] It is understood that although the present application describes the inventions involved in the above 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, without departing from the spirit of the invention described in the present application, various equivalent changes may be made to the technical features included in the inventions involved therein, and these changes should all fall within the scope of the invention.

Claims

1. A promoter comprising the nucleotide sequence shown in SEQ ID NO:1 or a nucleotide sequence having at least 90% identity with SEQ ID NO:1; preferably, 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; more preferably, the promoter consists 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; Preferably, the microorganism is a bacterium or a fungus; more preferably, the microorganism is Escherichia coli, Klebsiella pneumoniae, Corynebacterium glutamicum, Bacillus subtilis or yeast.

5. 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 claim 4 in regulating the expression intensity of a target gene, biocatalysis or biofermentation.

6. The method according to claim 5, wherein the regulating the expression intensity of the target gene is to enhance the expression intensity of the target gene.

7. The use according to claim 5 or 6, wherein the target gene is a gene encoding a protein; preferably, the protein is a related enzyme in an amino acid metabolic pathway; more preferably, the protein is a related enzyme in a branched-chain amino acid metabolic pathway; most preferably, the related enzyme is α-isopropylmalate synthase, β-isopropylmalate dehydrogenase, α-isopropylmalate isomerase, leucine dehydrogenase, acetohydroxyacid synthase, hydroxyacid reductoisomerase, dihydroxyacid 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.

8. The use according to claim 7, wherein the target gene is leuA, leuB, leuCD, leuDH, bcd, ilvBN, ilvC, ilvD, ilvE, brnFE, yeaS, pntAB, coaA, rocG, fxpk, leuE or tyrB gene; preferably, the target gene is leuA, leuB, leuCD, leuDH or tyrB gene.

9. The use as claimed in claim 5, wherein The biocatalysis is the catalysis of a substrate by an enzyme, and the expression of the enzyme is regulated by the promoter according to claim 1; and / or The biological fermentation is fermentation of amino acids, preferably fermentation of branched-chain amino acids.

10. A method for increasing amino acid production, comprising using the microorganism according to claim 4; preferably, the amino acid is a branched-chain amino acid.

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