CN117487824A - Artificial expression regulatory element and application thereof - Google Patents
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- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 208000009305 pseudorabies Diseases 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000003584 silencer Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- 101150099081 trpF gene Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
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Abstract
The invention discloses an artificial expression regulatory element and application thereof. In particular, the present invention relates to polynucleotides having expression control activity, transcription expression cassettes, recombinant expression vectors, recombinant host cells containing the foregoing polynucleotides, as well as methods for enhancing expression of a target gene, methods for producing a protein, and methods for producing an amino acid. The nucleotide sequence of the polynucleotide with the expression regulation activity is shown as SEQ ID NO:3 is shown in SEQ ID NO:1, and a variant of the polynucleotide of SEQ ID NO: compared with the polynucleotide of the sequence shown in 1, the expression regulation activity of the mutant is obviously enhanced, and the stable and efficient expression of the target gene can be promoted, so that the amino acid production efficiency of the recombinant strain is improved, and the recombinant strain has higher application value.
Description
Technical Field
The invention belongs to the technical field of biotechnology and genetic engineering, and in particular relates to a polynucleotide with expression regulation activity, a transcription expression cassette containing the polynucleotide with the expression regulation activity, a recombinant expression vector, a recombinant host cell, a method for enhancing target gene expression, and a method for preparing amino acid, especially hydroxyproline.
Background
trans-4-hydroxy-L-proline (abbreviated as hydroxyproline) is commonly found in nature and is widely used in the fields of medicine, food additives, animal feed, cosmetology industry and the like, and is mainly used for synthesizing side chains of carbapenem (third generation, last line of antibiotic) antibiotics at present due to high price. The third-generation antibiotics are atypical beta-lactam antibiotics with the widest antibacterial spectrum, and have the characteristics of wide antibacterial spectrum and strong antibacterial activity, and the global total market exceeds 30 hundred million dollars.
At present, the hydroxyproline is produced by a microbial fermentation method, and the L-proline is mainly converted into the hydroxyproline by exogenously adding L-proline under the action of proline-4-hydroxylase. Compared with exogenous L-proline production of trans-4-hydroxy-L-proline, direct endogenous synthesis of L-proline by glucose and other raw materials has obvious cost advantage, and enhancing the expression of Glutamate kinase ProB (Glutamate-5-kinase, genBank number: NP-414777.1) for relieving feedback inhibition of L-proline is an important means for enhancing endogenous L-proline synthesis of strains. ProB, which is mainly used for relieving feedback inhibition, is mainly studied, however, in industrial application, the problem that unstable plasmids are easy to lose is often caused by over-expression of plasmids, production cost is increased due to the addition of various antibiotics and the like for preventing the loss of plasmids, and the over-expression of the plasmids by adopting a strong expression element on a genome has the advantage of bacterial strain stability, however, elements capable of efficiently expressing the ProB in escherichia coli are still lacking at present.
Disclosure of Invention
The invention aims to solve the problem that plasmids are easy to lose in the production process of hydroxyproline, and obtains a promoter capable of efficiently and stably expressing ProB on a genome by modifying a promoter derived from corynebacterium glutamicum, thereby completing the invention on the basis. The invention comprises the following steps:
in a first aspect, there is provided a polynucleotide having expression regulatory activity, wherein the polynucleotide is selected from any one of the following (i) - (iv):
(i) Comprising a nucleotide sequence as shown in SEQ ID NO. 3;
(ii) A reverse complement comprising the nucleotide sequence shown as SEQ ID NO. 3;
(iii) A reverse complement of a sequence capable of hybridizing to the nucleotide sequence shown in (i) or (ii) under high stringency hybridization conditions or very high stringency hybridization conditions;
(iv) Has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the nucleotide sequence shown in (i) or (ii).
In a second aspect, there is provided a transcriptional expression cassette comprising a polynucleotide having expression regulatory activity according to the first aspect; optionally, the transcriptional expression cassette further comprises a protein-encoding gene operably linked to the polynucleotide having expression regulatory activity.
In a third aspect, there is provided a recombinant expression vector comprising the polynucleotide having expression control activity of the first aspect, or the transcriptional expression cassette of the second aspect.
In a fourth aspect, there is provided a recombinant microbial host cell comprising the transcriptional expression cassette of the second aspect, or the recombinant expression vector of the third aspect.
In a preferred embodiment of the invention, the recombinant microbial host cell is derived from the genus Escherichia, corynebacterium, brevibacterium, arthrobacter or Microbacterium; preferably, the host cell is E.coli or Corynebacterium glutamicum.
In a fifth aspect, there is provided the use of a polynucleotide having expression regulatory activity according to the first aspect, a transcription expression cassette according to the second aspect, a recombinant expression vector according to the third aspect, or a recombinant host cell according to the fourth aspect in at least one of:
(i) Preparing a reagent or a kit for enhancing the transcription level of a gene;
(ii) Preparing a protein, or preparing a reagent or kit for preparing a protein; preferably, the protein is an enzyme involved in amino acid synthesis; further preferably, the protein is the glutamate kinase ProB; more preferably, the glutamate kinase ProB is a glutamate kinase that releases feedback inhibition by L-proline; optionally, the Glutamate kinase ProB is obtained by introducing D107A amino acid mutation which can relieve feedback inhibition of L-proline into a coding gene of Glutamate kinase ProB (Glutamate-5-kinase, genBank number: NP 414777.1);
(iii) Producing amino acids; preferably, the amino acid is proline, hydroxyproline.
In a sixth aspect, there is provided a method of enhancing expression of a target gene, wherein the method comprises the step of operably linking a polynucleotide having expression regulatory activity according to the first aspect to the target gene.
In a seventh aspect, there is provided a method of producing a protein, wherein the transcriptional expression cassette of the second aspect, the recombinant expression vector of the third aspect, or the recombinant host cell of the fourth aspect is selected to express the protein; preferably, the protein is an enzyme involved in amino acid synthesis; further preferably, the protein is the glutamate kinase ProB; more preferably, the glutamate kinase ProB is a glutamate kinase that releases feedback inhibition by L-proline; alternatively, the glutamate kinase ProB is a D107A amino acid mutation which can release the feedback inhibition of L-proline and is introduced into a coding gene of the glutamate kinase ProB with the GenBank number of NP 414777.1.
In an eighth aspect, there is provided a method for producing an amino acid, wherein the polynucleotide of the first aspect, the transcriptional expression cassette of the second aspect, the recombinant expression vector of the third aspect, or the recombinant host cell of the fourth aspect expresses an enzyme involved in amino acid synthesis, and the amino acid is produced in the presence of the enzyme involved in amino acid synthesis, optionally the method further comprising the step of isolating or purifying the amino acid;
preferably, the amino acid is proline or hydroxyproline, and the enzyme related to amino acid synthesis is glutamate kinase ProB. Further preferably, the glutamate kinase ProB is a glutamate kinase which releases feedback inhibition by L-proline; alternatively, the glutamate kinase ProB is a D107A amino acid mutation which can release the feedback inhibition of L-proline and is introduced into a coding gene of the glutamate kinase ProB with the GenBank number of NP 414777.1.
The invention has the beneficial effects that:
in one embodiment, the present disclosure provides polynucleotides having expression modulating activity, the nucleotide sequences of which are set forth in SEQ ID NOs: 3, a mutant of the expression regulatory element of the sod gene derived from Corynebacterium glutamicum having significantly improved expression regulatory activity. Compared with a wild type sod gene promoter, the expression regulation activity of the mutant is obviously improved, and the mutant is operably connected with a target gene, so that the expression intensity of the target gene can be obviously improved, the stability of a genome can not be damaged, and the target gene can be stably and efficiently expressed, so that downstream products can be stably and efficiently produced.
In another embodiment, the present disclosure provides a method for producing amino acids, which can increase the expression of enzymes involved in amino acid synthesis by using the polynucleotide having the expression control activity, thereby stably and efficiently producing amino acids. When used for trans-4-hydroxy-L-proline or proline production, stable high yields of trans-4-hydroxy-L-proline or proline can be obtained.
Drawings
FIG. 1 shows a plasmid map of pSB4K5-rfp-1 plasmid.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Definition and description:
the term "polynucleotide" in the present invention refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments or may be an integral part of a larger nucleotide sequence structure, derived from nucleotide sequences that are separated at least once in number or concentration, and capable of identifying, manipulating and recovering sequences and their constituent nucleotide sequences by standard molecular biological methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (individual fragments or whole fragments), or may be a component or constituent of a larger nucleotide structure, such as an expression vector or polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.
The term "mutation" in the present invention refers to a nucleotide that comprises a mutation at one or more (e.g., several) positions of a polynucleotide, and retains the promoter activity of the polynucleotide. Wherein, the mutation (comprising, substituting, inserting and/or deleting) refers to the substitution, wherein, the substitution refers to the substitution of the nucleotide occupying one position with different nucleotides. Deletions refer to the removal of a nucleotide occupying a position. Insertion refers to the addition of nucleotides following the nucleotides that abut and immediately occupy the position.
The terms "sequence identity" and "percent identity" in the present invention refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides may be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides that contain the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides that contain a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or by deleting nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or both polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or by deleting amino acids (i.e., amino acid insertions or amino acid deletions in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
The term "complementary" in the present invention refers to hybridization or base pairing between nucleotides or nucleotides, such as between two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleotide being sequenced or amplified, etc.
The term "high stringency conditions" according to the invention means that for probes of at least 100 nucleotides in length, prehybridization and hybridization is performed at 42 ℃ in 5X SSPE (saline sodium phosphate EDTA), 0.3% sds, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard southern blotting procedures for 12 to 24 hours. Finally, the carrier material was washed three times, 15 minutes each, with 2 XSSC, 0.2% SDS at 65 ℃.
The term "very high stringency conditions" according to the invention means that for probes of at least 100 nucleotides in length, prehybridization and hybridization is performed at 42 ℃ in 5X SSPE (saline sodium phosphate EDTA), 0.3% sds, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard southern blotting procedures for 12 to 24 hours. Finally, the carrier material was washed three times, 15 minutes each, with 2 XSSC, 0.2% SDS at 70 ℃.
The term "promoter" as used herein refers to a nucleic acid molecule, typically located upstream of the coding sequence of the gene of interest, which provides a recognition site for RNA polymerase and is located 5' upstream of the transcription initiation site of mRNA. It is a nucleic acid sequence that is not translated, and RNA polymerase, when bound to this nucleic acid sequence, initiates transcription of the gene of interest. In ribonucleic acid (RNA) synthesis, a promoter can interact with a transcription factor that regulates gene transcription, controlling the start time and extent of gene expression (transcription), including core promoter and regulatory regions, like "on-off", to determine the activity of the gene and thus which protein the cell begins to produce.
The term "RBS" in the present invention means a ribosome binding site (ribosomebinding site, abbreviated RBS) which means that a consensus sequence consisting of 4 to 9 nucleotides, AGGAGG-, exists at about 8 to 13 nucleotides upstream of the initial AUG of mRNA, and can be precisely recognized by 16SrRNA through base complementation.
The term "RBS spacer" in the present invention refers to a nucleic acid sequence located in the promoter region of a prokaryote, a nucleotide sequence between RBS and the initiation codon.
The term "start codon" in the present invention has a definition well known to those skilled in the art and refers to a codon at the start site of protein synthesis, typically including ATG, GTG, TTG and ATA.
The term "expression control element" according to the invention has the definition known to the person skilled in the art and refers to a non-coding region DNA sequence located upstream of the start codon of a gene, including the sequences of the promoter, RBS and the spacer of the start codon and RBS, etc., capable of initiating transcription and expression of a downstream gene.
The term "expression" in the present invention includes any step involving RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term "protein-encoding gene" in the present invention refers to a synthetic DNA molecule capable of directing protein synthesis by a certain rule, and the process of protein-encoding gene directing protein synthesis generally includes a transcription process using double-stranded DNA as a template and a translation process using mRNA as a template. The protein Coding gene contains a CDS Sequence (Coding Sequence) capable of directing the production of mRNA encoding the protein. Protein-encoding genes include, but are not limited to, those encoding enzymes involved in the synthesis of amino acids, and in some embodiments, the protein-encoding genes are involved in encoding enzymes involved in the synthesis of L-proline, hydroxyproline. In some embodiments, the protein-encoding gene is involved in encoding enzymes involved in the synthesis of L-proline, hydroxyproline and derivatives thereof, such as gamma-glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, proline 4-hydroxylase, proline 3-hydroxylase, proline transporter, and the like. The polynucleotide with the expression regulation activity can be suitable for regulating and controlling the expression of target genes, and realizes the efficient production of target products.
The term "transcriptional expression cassette" in the present invention refers to an expression element comprising a transcriptional regulatory element and a target gene, which is used to regulate the expression of the target gene. In the present invention, the transcription regulatory element includes a promoter, and may further include an enhancer, a silencer, an insulator, and the like. In the present invention, the target gene is specifically a protein-encoding gene. By "operably linked" a target gene to a polynucleotide is meant that the polynucleotide having expression control activity is functionally linked to the target gene to initiate and mediate transcription of the target gene in any manner described by one of skill in the art.
The term "target gene" in the present invention relates to a gene of any one of which is linked to a polynucleotide having an expression control activity in the present disclosure to control the transcription level thereof.
In some embodiments, the target gene refers to a gene encoding a target protein in a microorganism. Alternatively, the target gene is a gene encoding an enzyme involved in biosynthesis of an amino acid, a gene encoding an enzyme involved in reducing power, a gene encoding an enzyme involved in glycolysis or TCA cycle, or a gene encoding an enzyme involved in release of a target compound, or the like. In some specific embodiments, the gene of interest may be a gene encoding gamma-glutamyl kinase, a gene encoding glutamate semialdehyde dehydrogenase, a gene encoding pyrroline-5-carboxylate reductase, a gene encoding proline 4-hydroxylase, a gene encoding proline 3-hydroxylase, a gene encoding proline transporter, and the like.
The term "vector" in the present invention refers to a DNA construct comprising a DNA sequence operably linked to suitable control sequences to express a gene of interest in a suitable host. "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors may include, for example, vectors comprising i) a collection of genetic elements, such as promoters and enhancers, that have a regulatory effect on gene expression; ii) a structural or coding sequence transcribed into mRNA and translated into protein; and iii) transcriptional subunits of appropriate transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not critical and any vector may be used, including plasmids, viruses, phages and transposons. Possible vectors for use in the present invention include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, chicken pox, baculovirus, SV40, and pseudorabies.
The term "amino acid" or "L-amino acid" in the present invention generally refers to the basic building block of a protein in which amino and carboxyl groups are bound to the same carbon atom. For example, the amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, 5-aminolevulinic acid or a derivative of an amino acid of any of the foregoing. In addition, the amino acid may be other kinds of amino acids in the art.
The term "microbial host cell" in the present invention means any microbial cell type that is easy to transform, transfect, transduce, etc., with a transcription initiation element or expression vector comprising a polynucleotide of the present invention. The term "recombinant microbial host cell" encompasses host cells which differ from the parent cell after the introduction of a transcription initiation element or recombinant expression vector, in particular by transformation.
The term "transformation" in the present invention has the meaning commonly understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The transformation method includes any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate (CaPO) 4 ) Precipitation method, calcium chloride (CaCl) 2 ) Precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, and lithium acetate-DMSO.
In one embodiment, the host cell refers to a microorganism suitable for fermentative production of amino acids, e.g., corynebacterium, brevibacterium, arthrobacter, microbacterium or Escherichia. Preferably, the host cell is Escherichia coli or Corynebacterium glutamicum.
The cultivation of the host cell of the present invention may be carried out according to a conventional method in the art, including but not limited to well plate cultivation, shake flask cultivation, batch cultivation, continuous cultivation, fed-batch cultivation, etc., and various cultivation conditions such as temperature, time, pH value of the medium, etc., may be appropriately adjusted according to the actual situation.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Example 1: evaluation of the potential of Corynebacterium glutamicum-derived expression regulatory elements for the expression of ProB in E.coli
In bacteria, the expression control sequences and the N-terminal coding region of the genes are key regions affecting gene expression. The invention adopts a method of sequentially connecting an expression control region of a gene, a 180bp coding region at the N end of the gene, a flexible linker and a red fluorescent protein gene rfp, and evaluates the expression intensity of the expression control sequence of a proB gene based on fluorescence intensity.
Previous studies have unexpectedly found that the expression regulatory element of the sod gene derived from Corynebacterium glutamicum has a higher expression level in Escherichia coli. The invention firstly uses the known strong expression element P in colibacillus trp The promoter was used as a control to evaluate the potential of the expression regulatory element of the sod gene derived from Corynebacterium glutamicum to express ProB.
Characterization of 2 expression intensities vectors were constructed as follows: based on pSB4K5 plasmid skeleton, the N end 60 amino acids of proB gene, a linker connecting peptide and red fluorescent protein gene are expressed by the selected expression regulatory element. pSB4K5 plasmid backbone DNA fragment 1 was amplified using pSB4K5-I52002 (GenBank: EU 496099.1) plasmid as a template and pSB4K5-F and pSB4K5-R as primers. According to published genome sequence of escherichia coli MG1655 (GenBank number: NC-000913) and annotation information of proB gene, designing a primer proB180-F/R, taking the escherichia coli MG1655 genome as a template, and obtaining a DNA fragment 2 with 180bp of the N end of the proB gene through PCR amplification. The pEC-XK99E-rfp-1 plasmid in the patent CN112695036B is used as a template, rfp-F and rfp-R are used as primers, and a linker connecting peptide and a red fluorescent protein gene fragment DNA fragment 3 are amplified. The expression regulatory element DNA fragment 4 (SEQ ID NO. 1) of the sod gene was amplified using the Corynebacterium glutamicum ATCC13032 genome (GenBank: BA 000036) as a template and sod-F and sod-R as primers. The inclusion of P was amplified using plasmid pTc-BA (J Biosci bioeng.2018Oct;126 (4): 470-477.) as template and trpF and trp-R as primers trp High-efficiency expression element fragment 5 (SEQ ID NO. 2) of the promoter and proB gene natural RBS. The DNA fragments 1-3 and the DNA fragment 4 are cloned and connected through a one-step recombination kit of the nuuzan to obtain the pSB4K5-rfp-1 characterization vector, and the plasmid map is shown in figure 1. The above DNA fragment 1The-3 and DNA fragment 5 were cloned and ligated by means of a one-step recombination kit of Norflu to obtain pSB4K5-rfp-control characterization vector. The primer sequences used in this example are shown in Table 1.
TABLE 1
To characterize the expression regulatory elements (SEQ ID NO: 1) of the corynebacterium glutamicum-derived sod gene, the constructed pSB4K5-rfp-1 and pSB4K5-rfp-control were transformed into E.coli MG1655, respectively, to obtain E.coli MG1655 (pSB 4K 5-rfp-1) and E.coli MG1655 (pSB 4K 5-rfp-control) strains. Strains obtained from LB plates were inoculated with toothpicks into 96-well plates containing 200. Mu.l of FJH liquid medium per well, 3 strains per well were parallel, the rotation speed of the shaking table of the well plates was 800rpm, and after culturing at 33℃for 21 hours, the fluorescence intensity (excitation wavelength: 560nm, emission wavelength: 607 nm) of the strains was measured by an enzyme-labeled instrument, and the expression intensity was characterized by the fluorescence intensity. FJH medium: glucose 10g/L; fish meal peptone 8g/L; 5g/L of ammonium sulfate; 1g/L of dipotassium hydrogen phosphate; sodium chloride 2g/L; 0.5g/L magnesium sulfate; ferrous sulfate 0.278g/L; 0.015g/L of calcium chloride; MOPS 40g/L, pH7.0. As shown in Table 2, the fluorescence intensity of the expression regulatory element of the sod gene derived from Corynebacterium glutamicum was improved by 79% as compared with the control, indicating that the expression regulatory element can be used for expression of ProB.
TABLE 2
| Strain | Fluorescence intensity (RFU/OD) 600 ) |
| E.coli MG1655(pSB4K5-rfp-control) | 1308±365 |
| E.coli MG1655(pSB4K5-rfp-1) | 2339±155 |
Example 2 engineering expression regulatory sequences to further increase the expression intensity of the element
This example describes the use of pSB4K5-rfp-1 plasmid(the 162 th to 171 th sequences underlined as SEQ ID NO:1 are the sequences of the modified region; bold ATG is the start codon of the proB gene) modified to +.>The polynucleotide sequence with expression regulation activity obtained after transformation is shown as a sequence SEQ ID NO: 3.
The concrete construction is as follows: the pSB4K5-rfp-1 plasmid is used as a template, RBS-1 and RBS-2 are used as primers, and fragments of the transformation region are amplified respectively. The plasmid backbone was amplified using pSB4K5-rfp-1 plasmid as template and RBS-3 and RBS-4 as primers. The 2 fragments are cloned and connected through a one-step recombination kit of the nupraise to obtain the pSB4K5-rfp-2 characterization vector. The primer sequences used in this example are shown in Table 3.
TABLE 3 Table 3
Using the same method as in example 1, SEQ ID NO:3, and an expression regulatory element shown in 3. The measurement results are shown in Table 4, and the sequences of SEQ ID NO:1, compared to the expression regulatory element shown in SEQ ID NO:3, the fluorescence intensity of the expression regulatory element is improved by 12.9 times, which indicates that the expression regulatory element can be used for efficiently expressing ProB.
TABLE 4 Table 4
| Strain | Fluorescence intensity (RFU/OD) 600 ) |
| E.coli MG1655(pSB4K5-rfp-1) | 2339±155 |
| E.coli MG1655(pSB4K5-rfp-2) | 32588±2660 |
EXAMPLE 3 production of trans-4-hydroxy-L-proline Using engineered ProB expression regulatory elements
Firstly, constructing a basic strain for producing trans-4-hydroxy-L-proline. By adopting a conventional molecular method, the coding gene of proline dehydrogenase PutA (proline dehydrogenase, genBank number: NP-415534.1) is knocked out on the genome of Escherichia coli MG1655, and simultaneously, D107A amino acid mutation (codon from GAT to GCT) capable of relieving feedback inhibition by L-proline is introduced on the coding gene of Glutamate kinase ProB (Glutamate-5-kinase, genBank number: NP-414777.1), so as to obtain EcP0 strain. Total gene synthesis P trp A promoter, expressing a fragment of the expression cassette sequence (SEQ ID NO. 4) of the proline 4-hydroxylase derived from dactylotheca RH 1; pTrc-1 and pTrc-2 primers are adopted, pTrc99a plasmid (GenBank number: U13872.1) is used as a template to amplify a plasmid skeleton, and the above 2 fragments are cloned and connected by a one-step recombination kit of Noruzhan to obtain pTrc99a-P trp Dap4h plasmid. pTrc99a-P trp Introducing the dap4h plasmid into EcP strain to obtain trans-4-hydroxy-L-proline production base strain EcP0 (pTrc 99 a-P) trp -dap4h)。
Reference (Sci Rep.2017Nov 30;7 (1): 16624.)The expression regulatory element shown in SEQ ID NO.3 was inserted upstream of the initiation codon of the proB gene of strain EcP 0. The preparation of the editing template is as follows: pSB4K5 plasmid backbone DNA fragment 1 was amplified using pSB4K5-I52002 (GenBank: EU 496099.1) plasmid as a template and pSB4K5-F and pSB4K5-R as primers in example 1; amplifying an upstream homology arm DNA fragment 2 by taking a EcP strain genome as a template and proB-1 and proB-2 as primers; amplifying the antibiotic marked DNA fragment 3 by taking pACYC184-M-crt plasmid in the literature as a template and cat-1 and cat-2 as primers; amplifying a DNA fragment 4 of the expression regulatory element shown in SEQ ID NO.3 by taking a plasmid pSB4K5-rfp-2 as a template and sod-3 and sod-4 as primers; the genome of the EcP strain is used as a template, and proB-3 and proB-4 are used as primers to amplify the downstream homology arm DNA fragment 5. The above 5 fragments were cloned and ligated by means of a one-step recombination kit of Norflu to obtain a plasmid containing the edited template. And amplifying the DNA fragment of the editing template, which is introduced with the expression regulatory element shown in SEQ ID NO.3, by using the plasmid containing the editing template as the template and pSB4K5-1 and pSB4K5-2 as primers. Using the above-obtained edited template DNA fragment and the procedure of reference (Sci Rep.2017Nov 30;7 (1): 16624.), a mutant strain EcP was obtained in which the expression control element shown in SEQ ID NO.3 was inserted upstream of the initiation codon of the proB gene of the EcP strain, and the expression control element upstream of the proB gene and the coding gene sequence in this strain were as shown in SEQ ID NO. 5. pTrc99a-P trp Introducing the dap4h plasmid into EcP strain to obtain an engineered trans-4-hydroxy-L-proline producing strain EcP1 (pTrc 99 a-P) trp Dap4 h). The primer sequences used in this example are shown in Table 5.
TABLE 5
Evaluation of expression regulatory element pair trans-4-hydroxy-L-proline for modification of proB Gene on genome by Orifice fermentationInfluence of acid production. Seed culture medium: 5g/L of yeast extract; 10g/L tryptone; sodium chloride 10g/L. Fermentation medium: glucose 10g/L; fish meal peptone 8g/L; 5g/L of ammonium sulfate; 1g/L of dipotassium hydrogen phosphate; sodium chloride 2g/L; 0.5g/L magnesium sulfate; ferrous sulfate 0.278g/L; 0.015g/L of calcium chloride; MOPS 40g/L, pH7.0. 100mg/L ampicillin was added during the strain cultivation. First, the strains were inoculated into liquid seed medium and cultured for 8 hours, respectively, and the cultures were inoculated as seeds into 24-well plates each containing 800. Mu.l of fermentation medium, initial OD 600 The culture was carried out at 33℃for 18 hours under control of about 0.1 at a rotation speed of 800rpm in a shaking table of an orifice plate, 3 strains each were in parallel, and the production of trans-4-hydroxy-L-proline and L-proline was examined after the completion of fermentation. The detection method of the trans-4-hydroxy-L-proline refers to national standard GB/T9695.23-2008. The results are shown in Table 6, and compared with the control strain, ecP1 (pTrc 99a-P trp The hydroxyproline yield of the dap4 h) strain can be increased by a factor of 3.5.
TABLE 6
Example 4.5L tank fermentation to produce trans-4-hydroxy-L-proline
EcP1(pTrc99a-P trp Dap4 h) strain was subjected to a 5L fermenter test for productivity. Seed culture medium: yeast extract, 5g/L; tryptone, 10g/L; sodium chloride, 10g/L. Fermentation tank medium: glucose, 20g/L; yeast powder, 5g/L; 10g/L of ammonium sulfate, 10g/L of monopotassium phosphate; magnesium sulfate, 0.5g/L; ferrous sulfate, 0.2g/L. 100mg/L ampicillin is added into the seeds of the strain and a fermentation medium, and a proper amount of defoamer is added into the fermentation medium. 100mL of seed liquid cultured overnight is inoculated into 2L of fermentation medium, the temperature is controlled to 33 ℃, the pH is controlled to 6.5 by adopting ammonia water, the dissolved oxygen is controlled to 30% by introducing sterile air, and the concentration of glucose is controlled to about 10g/L by feeding 80% glucose。OD 600 Detection using a spectrophotometer, detection of trans-4-hydroxy-L-proline was as described in example 3.EcP1 (pTrc 99 a-P) trp The results of fermentation in a 5L tank of the strain dap4 h) are shown in Table 7, the yield of trans-4-hydroxy-L-proline obtained by fermenting for 30 hours reaches 39.4g/L, and the production intensity of trans-4-hydroxy-L-proline is 1.313g/L/h.
TABLE 7
As can be seen from the above results, ecP1 (pTrc 99a-P trp The dap4 h) strain can efficiently produce trans-4-hydroxy-L-proline, which shows that the expression regulation element shown in SEQ ID NO.3 can be used for enhancing the expression of ProB by modifying the genome level, enhancing the synthesis of precursor L-proline and realizing the one-step fermentation production of trans-4-hydroxy-L-proline by taking glucose as a main raw material. Therefore, the expression regulatory element shown in SEQ ID NO.3, the expression cassette for enhancing the expression of ProB by the expression regulatory element shown in SEQ ID NO.3 and the trans-4-hydroxy-L-proline production strain transformed by the elements have very good industrialized application prospects.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A polynucleotide having expression control activity, wherein the polynucleotide is selected from any one of the following (i) to (iv):
(i) Comprising a nucleotide sequence as shown in SEQ ID NO. 3;
(ii) A reverse complement comprising the nucleotide sequence shown as SEQ ID NO. 3;
(iii) A reverse complement of a sequence capable of hybridizing to the nucleotide sequence shown in (i) or (ii) under high stringency hybridization conditions or very high stringency hybridization conditions;
(iv) Has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the nucleotide sequence shown in (i) or (ii).
2. A transcriptional expression cassette comprising the polynucleotide of claim 1 having expression regulatory activity; optionally, the transcriptional expression cassette further comprises a protein-encoding gene operably linked to the polynucleotide having expression regulatory activity.
3. A recombinant expression vector comprising the polynucleotide of claim 1 having expression control activity or the transcriptional expression cassette of claim 2.
4. A recombinant microbial host cell comprising the transcriptional expression cassette of claim 2 or the recombinant expression vector of claim 3.
5. The host cell of claim 4, wherein the recombinant microbial host cell is derived from the genera Escherichia, corynebacterium, brevibacterium, arthrobacter, microbacterium; preferably, the host cells are E.coli and Corynebacterium glutamicum.
6. Use of the polynucleotide having expression regulatory activity of claim 1, the transcriptional expression cassette of claim 2, the recombinant expression vector of claim 3, or the recombinant host cell of claim 4 or 5 in at least one of:
(i) Preparing a reagent or a kit for enhancing the transcription level of a gene;
(ii) Preparing a protein, or preparing a reagent or kit for preparing a protein; preferably, the protein is an enzyme involved in the synthesis of amino acids; further preferably, the protein is the glutamate kinase ProB; more preferably, the glutamate kinase ProB is a glutamate kinase that releases feedback inhibition by L-proline; optionally, the glutamate kinase ProB is obtained by introducing D107A amino acid mutation which can relieve feedback inhibition of L-proline into a coding gene of the glutamate kinase ProB with the GenBank number of NP 414777.1;
(iii) Producing amino acids; preferably, the amino acid is proline, hydroxyproline.
7. A method of enhancing expression of a target gene, comprising the step of operably linking the polynucleotide having expression control activity of claim 1 to a target gene.
8. A method for producing a protein, characterized by selecting a polynucleotide having an expression regulatory activity according to claim 1, a transcription expression cassette according to claim 2, a recombinant expression vector according to claim 3, or a recombinant host cell according to claim 4 or 5 for expression of the protein; preferably, the protein is an enzyme involved in amino acid synthesis; further preferably, the protein is the glutamate kinase ProB; more preferably, the glutamate kinase ProB is a glutamate kinase that releases feedback inhibition by L-proline; alternatively, the glutamate kinase ProB is a D107A amino acid mutation which can release the feedback inhibition of L-proline and is introduced into a coding gene of the glutamate kinase ProB with the GenBank number of NP 414777.1.
9. A method for producing an amino acid, characterized in that a polynucleotide having an expression regulatory activity according to claim 1, a transcription expression cassette according to claim 2, a recombinant expression vector according to claim 3, or a recombinant host cell according to claim 4 or 5 is selected to express an enzyme involved in amino acid synthesis, and the amino acid is produced in the presence of the enzyme involved in amino acid synthesis, optionally the method further comprising a step of isolating or purifying the amino acid.
10. The method according to claim 9, wherein the amino acid is proline or hydroxyproline and the enzyme involved in amino acid synthesis is glutamate kinase ProB; further preferably, the glutamate kinase ProB is a glutamate kinase which releases feedback inhibition by L-proline; alternatively, the glutamate kinase ProB is a D107A amino acid mutation which can release the feedback inhibition of L-proline and is introduced into a coding gene of the glutamate kinase ProB with the GenBank number of NP 414777.1.
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