Artificial expression regulatory element and use thereof

Abstract

The application discloses an artificial expression regulatory element and application thereof. Specifically, the application relates to a polynucleotide with expression regulatory activity, a transcription expression cassette containing the polynucleotide, a recombinant expression vector, a recombinant host cell, and a method for enhancing expression of a target gene, a method for preparing protein and a method for producing amino acid. The nucleotide sequence of the polynucleotide with expression regulatory activity of the application is shown in SEQ ID NO: 3, and the polynucleotide is a mutant of the polynucleotide with the sequence of SEQ ID NO: 1. Compared with the polynucleotide with the sequence shown in SEQ ID NO: 1, the expression regulatory activity of the mutant is significantly enhanced, and the mutant can promote stable and efficient expression of a target gene, thereby improving the amino acid production efficiency of a recombinant strain, and having high application value.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and genetic engineering, specifically relating to a polynucleotide with expression regulatory activity, comprising a transcriptional expression cassette of the polynucleotide with expression regulatory activity, a recombinant expression vector, a recombinant host cell, a method for enhancing target gene expression, and a method for preparing amino acids, especially hydroxyproline. Background Technology

[0002] Trans-4-hydroxy-L-proline (hydroxyproline for short) is ubiquitous in nature and widely used in pharmaceuticals, food additives, animal feed, and the cosmetics industry. Due to its high price, it is currently mainly used as a side chain in the synthesis of carbapenems (third-generation antibiotics, the last line of defense against antibiotics). Third-generation antibiotics are the broadest-spectrum atypical β-lactam antibiotics, characterized by their broad antibacterial spectrum and strong antibacterial activity. The global market has exceeded $3 billion.

[0003] Currently, the production of hydroxyproline via microbial fermentation mainly involves the exogenous addition of L-proline, which is then converted to hydroxyproline by proline-4-hydroxylase. Compared to the production of trans-4-hydroxy-L-proline through exogenous L-proline addition, direct endogenous synthesis of L-proline from raw materials such as glucose has a significant cost advantage. Enhancing the expression of glutamate kinase ProB (Glutamate-5-kinase, GenBank ID: NP_414777.1), which relieves L-proline feedback inhibition, is an important means to strengthen the endogenous L-proline synthesis in bacterial strains. Existing studies mainly use plasmid overexpression to relieve feedback inhibition of ProB. However, in industrial applications, plasmid overexpression often suffers from instability and easy plasmid loss. The addition of various antibiotics to prevent plasmid loss increases production costs. Overexpression using strong expression elements in the genome offers advantages in strain stability. However, elements that can efficiently express ProB in Escherichia coli are still lacking. Summary of the Invention

[0004] This invention addresses the problem of plasmid loss during hydroxyproline production by modifying a promoter derived from Corynebacterium glutamicum to obtain a promoter capable of efficiently and stably expressing ProB on the genome. The invention is based on this modification.

[0005] In a first aspect, a polynucleotide with expression regulatory activity is provided, wherein the polynucleotide is selected from any one of (i)-(iv):

[0006] (i) Contains a nucleotide sequence as shown in SEQ ID NO.3;

[0007] (ii) A reverse complementary sequence comprising the nucleotide sequence shown in SEQ ID NO.3;

[0008] (iii) The reverse complementary sequence of a sequence that can hybridize with the nucleotide sequence shown in (i) or (ii) under high-strict hybridization conditions or very high-strict hybridization conditions;

[0009] (iv) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (i) or (ii).

[0010] In a second aspect, a transcription expression cassette is provided, comprising the polynucleotide with expression regulatory activity described in the first aspect; optionally, the transcription expression cassette further comprises a protein-coding gene, the protein-coding gene being operatively linked to the polynucleotide with expression regulatory activity.

[0011] Thirdly, a recombinant expression vector is provided, comprising the polynucleotide with expression regulatory activity described in the first aspect, or the transcription expression cassette described in the second aspect.

[0012] Fourthly, a recombinant microbial host cell is provided, comprising the transcription expression cassette described in the second aspect, or the recombinant expression vector described in the third aspect.

[0013] In a preferred embodiment of the present invention, the recombinant microbial host cell is derived from Escherichia coli, Corynebacterium, Brevibacterium, Arthrobacterium, or Microbacterium; preferably, the host cell is Escherichia coli or Corynebacterium glutamicum.

[0014] Fifthly, the use of the polynucleotide with expression regulatory activity described in the first aspect, the transcriptional expression cassette described in the second aspect, the recombinant expression vector described in the third aspect, or the recombinant host cell described in the fourth aspect in at least one of the following:

[0015] (i) Prepare reagents or kits for enhancing gene transcription levels;

[0016] (ii) Preparing a protein, or preparing a reagent or kit for preparing a protein; preferably, the protein is an enzyme related to amino acid synthesis; more preferably, the protein is glutamate kinase ProB; more preferably, the glutamate kinase ProB is a glutamate kinase that has relieved L-proline feedback inhibition; optionally, the glutamate kinase ProB is obtained by introducing a D107A amino acid mutation that can relieve L-proline feedback inhibition into the coding gene of glutamate kinase ProB (Glutamate-5-kinase, GenBank No.: NP_414777.1);

[0017] (iii) Producing amino acids; preferably, the amino acids are proline or hydroxyproline.

[0018] A sixth aspect provides a method for enhancing the expression of a target gene, wherein the method includes the step of operatively linking the polynucleotide having expression regulatory activity as described in the first aspect to the target gene.

[0019] In a seventh aspect, a method for preparing a protein is provided, wherein the protein is expressed using the transcriptional expression cassette described in the second aspect, the recombinant expression vector described in the third aspect, or the recombinant host cell described in the fourth aspect; preferably, the protein is an enzyme related to amino acid synthesis; more preferably, the protein is glutamate kinase ProB; more preferably, the glutamate kinase ProB is a glutamate kinase that has relieved L-proline feedback inhibition; optionally, the glutamate kinase ProB is obtained by introducing a D107A amino acid mutation that can relieve L-proline feedback inhibition into the encoding gene of glutamate kinase ProB with GenBank number NP_414777.1.

[0020] Eighthly, a method for producing amino acids is provided, wherein the polynucleotide described in the first aspect, the transcription expression cassette described in the second aspect, the recombinant expression vector described in the third aspect, or the recombinant host cell described in the fourth aspect is selected to express an enzyme related to amino acid synthesis, and the amino acids are produced in the presence of the enzyme related to amino acid synthesis. Optionally, the method further includes the step of isolating or purifying the amino acids.

[0021] Preferably, the amino acid is proline or hydroxyproline, and the enzyme related to the synthesis of the amino acid is glutamate kinase ProB. More preferably, the glutamate kinase ProB is a glutamate kinase that relieves L-proline feedback inhibition; optionally, the glutamate kinase ProB is obtained by introducing a D107A amino acid mutation that can relieve L-proline feedback inhibition into the coding gene of glutamate kinase ProB (GenBank accession number NP_414777.1).

[0022] The beneficial effects of this invention are:

[0023] In one embodiment, this disclosure provides a polynucleotide with expression regulatory activity, the nucleotide sequence of which is shown in SEQ ID NO: 3. This polynucleotide is a mutant of the expression regulatory element of the *Corynebacterium glutamicum*-derived *sod* gene, exhibiting significantly enhanced expression regulatory activity. Compared to the wild-type *sod* gene promoter, the mutant demonstrates significantly enhanced expression regulatory activity. Operable linking it to the target gene can significantly increase the expression intensity of the target gene without disrupting genome stability, enabling stable and efficient expression of the target gene, thereby achieving stable and efficient production of downstream products.

[0024] In another embodiment, this disclosure provides a method for producing amino acids, which utilizes the aforementioned polynucleotides with expression-regulating activity to enhance the expression of enzymes related to amino acid synthesis, thereby achieving stable and efficient production of amino acids. When used for the production of trans-4-hydroxy-L-proline or proline, stable and high-yield trans-4-hydroxy-L-proline or proline can be obtained. Attached Figure Description

[0025] Figure 1 Plasmid map of .pSB4K5-rfp-1 plasmid. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0027] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0028] Definitions and Explanations:

[0029] The term "polynucleotide" as used in this invention refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a single fragment or as a component of a larger nucleotide sequence structure derived from a nucleotide sequence isolated at least once in number or concentration, capable of being recognized, manipulated, and recovered using standard molecular biology methods (e.g., using cloning vectors). This also includes an RNA sequence (i.e., A, U, G, C) when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), where "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (single fragments or entire fragments), or it can be a component or part of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0030] The term "mutation" as used in this invention refers to the presence of a mutated nucleotide at one or more (e.g., several) positions of a polynucleotide while maintaining the promoter activity of the polynucleotide. Specifically, the term "mutation" (including substitution, insertion, and / or deletion) in this invention refers to substitution, whereby a nucleotide occupying a position is replaced with a different nucleotide. Deletion refers to the removal of a nucleotide occupying a position. Insertion refers to the addition of a nucleotide adjacent to and immediately following the nucleotide occupying the position.

[0031] In this invention, the terms "sequence identity" and "identity percentage" refer to the percentage of identical (i.e., the same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotide or polypeptide and scoring the number of positions in the aligned polynucleotide or polypeptide containing the same nucleotide or amino acid residues, comparing this to the number of positions in the aligned polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides may differ at a position, for example, by containing different nucleotides (i.e., substitutions or mutations) or deleted nucleotides (i.e., nucleotide insertions or deletions in one or two polynucleotides). Polypeptides may differ at a position, for example, by containing different amino acids (i.e., substitutions or mutations) or deleted amino acids (i.e., amino acid insertions or deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the identity percentage can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0032] The term "complementary" in this invention refers to hybridization or base pairing between nucleotides, such as between the 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.

[0033] The term "highly stringent conditions" in this invention refers to, for probes at least 100 nucleotides in length, following a standard DNA blotting procedure: pre-hybridization and hybridization at 42°C in 5X SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0034] The term "very stringent conditions" in this invention refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0035] In this invention, the term "promoter" refers to a nucleic acid molecule, typically located upstream of the coding sequence of a target gene, providing a recognition site for RNA polymerase and situated upstream of the mRNA transcription start site in the 5' direction. It is an untranslated nucleic acid sequence to which RNA polymerase binds, initiating transcription of the target gene. In ribonucleic acid (RNA) synthesis, promoters can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription). Containing a core promoter region and a regulatory region, promoters act like a "switch," determining gene activity and thus controlling which protein the cell begins to produce.

[0036] In this invention, the term "RBS" refers to the ribosome binding site, which is a shared sequence of 4 to 9 nucleotides, located approximately 8 to 13 nucleotides upstream of the initiation AUG of mRNA. This sequence can be precisely recognized by 16S rRNA through base complementarity.

[0037] In this invention, the term "RBS spacer" refers to a segment of nucleic acid sequence located in the promoter region of a prokaryote, which is a nucleotide sequence between the RBS and the start codon.

[0038] The term "start codon" in this invention has a definition known to those skilled in the art, referring to the codon at the protein synthesis initiation site, typically including ATG, GTG, TTG, and ATA.

[0039] The term "expression regulatory element" in this invention has a definition known to those skilled in the art, referring to a non-coding DNA sequence located upstream of the start codon of a gene, including sequences such as promoters, RBS, and the spacer region between the start codon and the RBS, which can initiate the transcription and expression of downstream genes.

[0040] The term "expression" in this invention includes any step involving RNA and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0041] In this invention, the term "protein-coding gene" refers to a DNA molecule capable of guiding protein synthesis according to certain rules. The process by which a protein-coding gene guides protein synthesis generally includes transcription using double-stranded DNA as a template and translation using mRNA as a template. Protein-coding genes contain a CDS (Coding Sequence) that guides the production of mRNA encoding proteins. Protein-coding genes include, but are not limited to, enzymes encoding amino acids. In some embodiments, protein-coding genes involve enzymes encoding L-proline and hydroxyproline. In some embodiments, protein-coding genes involve enzymes encoding L-proline, hydroxyproline, and their derivatives, such as γ-glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrrolidone-5-carboxylic acid reductase, proline 4-hydroxylase, proline 3-hydroxylase, proline transporters, etc. The polynucleotides of this invention with expression-regulating activity are suitable for regulating the expression of target genes, achieving efficient production of target products.

[0042] In this invention, the term "transcriptional expression cassette" refers to a class of expression elements that include a transcriptional regulatory element and a target gene, and that regulate the expression of the target gene using the transcriptional regulatory element. In this invention, the transcriptional regulatory element includes a promoter, and may also include enhancers, silencers, insulators, etc. In this invention, the target gene is specifically a protein-coding gene. "Operationally linked" between the target gene and a polynucleotide refers to the functional linking of a polynucleotide with expression regulatory activity to the target gene to initiate and mediate the transcription of the target gene. The operably linked method can be any method described by those skilled in the art.

[0043] The term "target gene" in this invention refers to any gene that is linked to a polynucleotide having expression regulatory activity as disclosed herein to regulate its transcriptional level.

[0044] In some embodiments, the target gene refers to a gene encoding a target protein in a microorganism. Optionally, the target gene may be a gene encoding an enzyme related to amino acid biosynthesis, an enzyme related to reducing power, an enzyme related to glycolysis or the TCA cycle, or an enzyme related to the release of a target compound, etc. In some specific embodiments, the target gene may be a gene encoding γ-glutamyl kinase, a gene encoding glutamate semialdehyde dehydrogenase, a gene encoding pyrroline-5-carboxylic acid reductase, a gene encoding proline 4-hydroxylase, a gene encoding proline 3-hydroxylase, a gene encoding a proline transporter, etc.

[0045] The term "vector" as used in this invention refers to a DNA construct containing a DNA sequence operatively linked to a suitable control sequence for expressing a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure for expressing, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence transcribed into mRNA and translated into a protein; and iii) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, and DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies.

[0046] The term "amino acid" or "L-amino acid" in this invention generally refers to the basic building block of proteins in which the amino and carboxyl groups are bonded to the same carbon atom. For example, an 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 derivatives of any of the above amino acids. Furthermore, an amino acid can also be any other type of amino acid in the art.

[0047] In this invention, the term "microbial host cell" refers to any type of microbial cell that is readily transformed, transfected, transduced, etc., using transcription initiation elements or expression vectors containing the polynucleotides of this invention. The term "recombinant microbial host cell" encompasses host cells that differ from parental cells after the introduction of transcription initiation elements or recombinant expression vectors; recombinant host cells are specifically achieved through transformation.

[0048] The term "transformation" in this invention has the meaning commonly understood by those skilled in the art, namely, the process of introducing exogenous DNA into a host. The methods of transformation include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0049] In one embodiment, the host cell refers to a microorganism suitable for fermentation production of amino acids, such as Corynebacterium, Brevibacterium, Arthrobacterium, Microbacterium, or Escherichia. Preferably, the host cell is Escherichia coli derived from Escherichia or Corynebacterium glutamicum derived from Corynebacterium.

[0050] The host cell culture of the present invention can be carried out according to conventional methods in the art, including but not limited to plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, and various culture conditions such as temperature, time and pH of the culture medium can be appropriately adjusted according to actual conditions.

[0051] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0052] Example 1: Evaluation of the potential of Corynebacterium glutamicum-derived expression regulatory elements for ProB expression in Escherichia coli

[0053] In bacteria, gene expression regulatory sequences and N-terminal coding regions are key areas affecting gene expression. This invention employs a method that sequentially links the gene expression regulatory region, the 180bp coding region at the N-terminus of the gene, a flexible linker, and a red fluorescent protein gene rfp, to evaluate the expression intensity of the proB gene expression regulatory sequence based on fluorescence intensity.

[0054] Previous studies unexpectedly revealed that expression regulatory elements of the SOD gene derived from Corynebacterium glutamicum are highly expressed in Escherichia coli. This invention first utilizes the known strongly expressed element P in Escherichia coli. trp The promoter was used as a control to evaluate the potential of ProB expression by the expression regulatory element of the sod gene derived from Corynebacterium glutamicum.

[0055] Two characterization vectors for expression intensity were constructed as follows: Based on the pSB4K5 plasmid backbone, the selected expression regulatory elements expressed the N-terminal 60 amino acids of the proB gene, a linker peptide, and the red fluorescent protein gene. Using the pSB4K5-I52002 plasmid (GenBank ID: EU496099.1) as a template, and pSB4K5-F and pSB4K5-R primers, the pSB4K5 plasmid backbone DNA fragment 1 was amplified. Based on the published E. coli MG1655 genome sequence (GenBank ID: NC_000913) and proB gene annotation information, primers proB180-F / R were designed. Using the E. coli MG1655 genome as a template, the N-terminal 180 bp DNA fragment 2 of the proB gene was amplified by PCR. Using the pEC-XK99E-rfp-1 plasmid from patent CN112695036B as a template, and using rfp-F and rfp-R as primers, the linker peptide and red fluorescent protein gene fragment DNA fragment 3 were amplified. Using the Corynebacterium glutamicum ATCC13032 genome (GenBank No.: BA000036) as a template, and using sod-F and sod-R as primers, the expression regulatory element DNA fragment 4 (SEQ ID NO.1) of the sod gene was amplified. Using the plasmid pTc-BA (J Biosci Bioeng. 2018 Oct; 126(4):470-477.) as a template, and using trpF and trp-R as primers, a fragment containing P was amplified. trp The promoter and high-efficiency expression element fragment 5 of the natural RBS of the proB gene (SEQ ID NO.2). DNA fragments 1-3 and DNA fragment 4 were cloned and ligated using a one-step recombination kit from Novizan to obtain the pSB4K5-rfp-1 characterization vector. The plasmid map is shown below. Figure 1 As shown in Table 1, DNA fragments 1-3 and DNA fragment 5 were cloned and ligated using a one-step recombination kit from Novizan to obtain the pSB4K5-rfp-control characterization vector. The primer sequences used in this example are shown in Table 1.

[0056] Table 1

[0057]

[0058]

[0059] To characterize the expression regulatory element of the sod gene (SEQ ID NO: 1) from Corynebacterium glutamicum, the constructed pSB4K5-rfp-1 and pSB4K5-rfp-control were transformed into E. coli MG1655, obtaining E. coli MG1655(pSB4K5-rfp-1) and E. coli MG1655(pSB4K5-rfp-control) strains, respectively. The strains obtained from LB agar plates were inoculated into 96-well plates containing 200 μl of FJH liquid medium per well using toothpicks, with three replicates per strain. The plates were shaken at 800 rpm and incubated at 33°C for 21 h. The fluorescence intensity of the strains was then detected using a microplate reader (excitation wavelength: 560 nm, emission wavelength: 607 nm), and the fluorescence intensity was used to characterize the expression intensity. FJH medium: glucose 10 g / L; fish meal peptone 8 g / L; ammonium sulfate 5 g / L; dipotassium hydrogen phosphate 1 g / L; sodium chloride 2 g / L; magnesium sulfate 0.5 g / L; ferrous sulfate 0.278 g / L; calcium chloride 0.015 g / L; MOPS 40 g / L, pH 7.0. The results are shown in Table 2. Compared with the control, the fluorescence intensity of the expression regulatory element of the *Corynebacterium glutamicum*-derived *sod* gene increased by 79%, indicating that this expression regulatory element can be used for ProB expression.

[0060] Table 2

[0061] E.coli MG1655(pSB4K5-rfp-control) 1308±365 E. coli MG1655(pSB4K5-rfp-1) 2339±155

[0062] Example 2. Modifying expression regulatory sequences to further enhance element expression intensity

[0063] This embodiment uses pSB4K5-rfp-1 plasmid... (The underlined part represents positions 162 to 171 of SEQ ID NO: 1, which is the modified region; bold ATG is the start codon of the proB gene.) Modified to The modified polynucleotide sequence with expression regulatory activity is shown in SEQ ID NO: 3.

[0064] The specific construction is as follows: Using pSB4K5-rfp-1 plasmid as a template, and RBS-1 and RBS-2 as primers, the modified region fragment was amplified. Using pSB4K5-rfp-1 plasmid as a template, and RBS-3 and RBS-4 as primers, the plasmid backbone was amplified. The above two fragments were cloned and ligated using Novizan's one-step recombination kit to obtain the pSB4K5-rfp-2 characterization vector. The primer sequences used in this example are shown in Table 3.

[0065] Table 3

[0066]

[0067] The expression regulatory element shown in SEQ ID NO: 3 was characterized using the same method as in Example 1. The measurement results are shown in Table 4. Compared with the expression regulatory element shown in SEQ ID NO: 1, the fluorescence intensity of the expression regulatory element shown in SEQ ID NO: 3 was increased by 12.9 times, indicating that this expression regulatory element can be used for efficient expression of ProB.

[0068] Table 4

[0069] E. coli MG1655(pSB4K5-rfp-1) 2339±155 E. coli MG1655(pSB4K5-rfp-2) 32588±2660

[0070] Example 3. Production of trans-4-hydroxy-L-proline using a modified ProB expression regulatory element

[0071] This invention first constructs a basic strain for producing trans-4-hydroxy-L-proline. Using conventional molecular methods, the gene encoding proline dehydrogenase PutA (GenBank ID: NP_415534.1) was knocked out in the genome of *E. coli* MG1655. Simultaneously, a D107A amino acid mutation (codon GCT from GAT) was introduced into the gene encoding glutamate kinase ProB (GenBank ID: NP_414777.1) to relieve L-proline feedback inhibition, resulting in strain EcP0. The entire genome of P is synthesized. trp The promoter was used to express the expression cassette sequence (SEQ ID NO.4) of proline 4-hydroxylase derived from *Dactylogyrus RH1*. Using pTrc-1 and pTrc-2 primers, the plasmid backbone was amplified using pTrc99a plasmid (GenBank ID: U13872.1) as a template. Both fragments were then cloned and ligated using a one-step recombination kit from Novizan to obtain pTrc99a-P. trp -dap4h plasmid. pTrc99a-P trp The -dap4h plasmid was introduced into the EcP0 strain to obtain the basic strain for trans-4-hydroxy-L-proline production, EcP0(pTrc99a-P). trp -dap4h).

[0072] The genome editing method described in the reference (Sci Rep. 2017 Nov 30; 7(1): 16624.) was used to insert the expression regulatory element shown in SEQ ID NO. 3 upstream of the start codon of the proB gene in the EcP0 strain. The template was prepared as follows: using the pSB4K5-I52002 plasmid (GenBank No.: EU496099.1) as a template, and using pSB4K5-F and pSB4K5-R from Example 1 as primers, the pSB4K5 plasmid backbone DNA fragment 1 was amplified; using the EcP0 strain genome as a template, and using proB-1 and proB-2 as primers, the upstream homologous arm DNA fragment 2 was amplified; using the pACYC184-M-crt plasmid from the literature as a template, and using cat-1 and cat-2 as primers, the antibiotic-labeled DNA fragment 3 was amplified; using the pSB4K5-rfp-2 plasmid as a template, and using sod-3 and sod-4 as primers, the DNA fragment 4 containing the expression regulatory element shown in SEQ ID NO.3 was amplified; using the EcP0 strain genome as a template, and using proB-3 and proB-4 as primers, the downstream homologous arm DNA fragment 5 was amplified. The above five fragments were cloned and ligated using the Novizan one-step recombination kit to obtain a plasmid containing the editing template. Using the plasmid containing the editing template as a template, and pSB4K5-1 and pSB4K5-2 as primers, the editing template DNA fragment containing the expression regulatory element shown in SEQ ID NO.3 was amplified. Using the editing template DNA fragment obtained above, and referring to the procedure in the literature (Sci Rep. 2017 Nov 30; 7(1):16624.), a mutant strain EcP1 was obtained, in which the expression regulatory element shown in SEQ ID NO.3 was inserted upstream of the start codon of the proB gene in strain EcP0. The expression regulatory element and coding gene sequence upstream of the proB gene in this strain are shown in SEQ ID NO.5. pTrc99a-P trp The -dap4h plasmid was introduced into the EcP1 strain to obtain the modified trans-4-hydroxy-L-proline producing strain EcP1(pTrc99a-P trp -dap4h). The primer sequences used in this embodiment are shown in Table 5.

[0073] Table 5

[0074]

[0075]

[0076] The effect of a genetically modified proB gene expression regulatory element on trans-4-hydroxy-L-proline production was evaluated using plate fermentation. Seed medium: yeast extract 5 g / L; tryptone 10 g / L; sodium chloride 10 g / L. Fermentation medium: glucose 10 g / L; fish meal peptone 8 g / L; ammonium sulfate 5 g / L; dipotassium hydrogen phosphate 1 g / L; sodium chloride 2 g / L; magnesium sulfate 0.5 g / L; ferrous sulfate 0.278 g / L; calcium chloride 0.015 g / L; MOPS 40 g / L, pH 7.0. 100 mg / L ampicillin was added during the bacterial culture process. First, the bacterial strain was inoculated into liquid seed medium and cultured for 8 h. The culture was then used as seed culture in 24-well plates containing 800 μl of fermentation medium per well. Initial OD... 600 The control temperature was approximately 0.1, and the culture was carried out at 33℃ for 18 hours. The shaking speed of the well plate was 800 rpm, and each strain was tested in triplicate. After fermentation, the yields of trans-4-hydroxy-L-proline and L-proline were measured. The detection method for trans-4-hydroxy-L-proline was based on the national standard GB / T 9695.23-2008. The results are shown in Table 6. Compared with the control strain, the modified EcP1(pTrc99a-P trp The hydroxyproline production of strain -dap4h can be increased by 3.5 times.

[0077] Table 6

[0078]

[0079]

[0080] Example 4.5L tank fermentation for the production of trans-4-hydroxy-L-proline

[0081] EcP1(pTrc99a-P trp The production performance of strain -dap4h was tested in a 5L fermenter. Seed culture medium: yeast extract, 5 g / L; tryptone, 10 g / L; sodium chloride, 10 g / L. Fermenter culture medium: glucose, 20 g / L; yeast extract, 5 g / L; ammonium sulfate, 10 g / L; potassium dihydrogen phosphate, 10 g / L; magnesium sulfate, 0.5 g / L; ferrous sulfate, 0.2 g / L. 100 mg / L ampicillin was added to both the seed culture and the fermentation medium, and an appropriate amount of antifoaming agent was added to the fermentation medium. 100 mL of overnight seed culture was inoculated into 2 L of fermentation medium. The temperature was controlled at 33℃, pH was controlled at 6.5 using ammonia, dissolved oxygen was controlled at 30% using sterile air, and 80% glucose was added to maintain a glucose concentration of approximately 10 g / L. OD 600 Detection was performed using a spectrophotometer. The detection of trans-4-hydroxy-L-proline was performed according to Example 3. EcP1(pTrc99a-P trpThe fermentation results of strain -dap4h) in a 5L tank are shown in Table 7. After 30 hours of fermentation, the yield of trans-4-hydroxy-L-proline reached 39.4 g / L, and the production intensity of trans-4-hydroxy-L-proline was 1.313 g / L / h.

[0082] Table 7

[0083]

[0084] The results above show that EcP1(pTrc99a-P trp The strain (-dap4h) can efficiently produce trans-4-hydroxy-L-proline, indicating that the expression regulatory element shown in SEQ ID NO.3 can be used to enhance ProB expression and strengthen the synthesis of precursor L-proline at the genomic level, enabling one-step fermentation production of trans-4-hydroxy-L-proline using glucose as the main raw material. Therefore, the expression regulatory element shown in SEQ ID NO.3, the expression cassette that enhances ProB expression using the expression regulatory element shown in SEQ ID NO.3, and the trans-4-hydroxy-L-proline producing strain modified with the above elements all have very good prospects for industrial application.

[0085] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polynucleotide with expression regulatory activity, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

3.

2. A transcription expression cassette, characterized in that, The transcription expression cassette comprises the polynucleotide with expression regulatory activity as described in claim 1.

3. The transcription expression cassette according to claim 2, characterized in that, The transcriptional expression cassette also contains a protein-coding gene, which is operatively linked to the polynucleotide having expression regulatory activity.

4. A recombinant expression vector, characterized in that, The expression vector comprises the polynucleotide with expression regulatory activity as described in claim 1, or the transcription expression cassette as described in claim 2 or 3.

5. A recombinant microbial host cell, characterized in that, The host cell comprises the transcriptional expression cassette of claim 2 or 3, or the recombinant expression vector of claim 4.

6. The host cell according to claim 5, characterized in that, The recombinant microbial host cells are derived from Escherichia coli, Corynebacterium, Brevibacterium, Arthrobacterium, and Microbacterium.

7. The host cell according to claim 6, characterized in that, The host cells are Escherichia coli and Corynebacterium glutamicum.

8. The use of the polynucleotide with expression regulatory activity according to claim 1, the transcription expression cassette according to claim 2 or 3, the recombinant expression vector according to claim 4, or the recombinant host cell according to any one of claims 5-7 in at least one of the following: (i) Prepare reagents or kits for enhancing the transcriptional level of the glutamate kinase ProB gene; (ii) Preparing a protein, or preparing a reagent or kit for preparing a protein, wherein the protein is a glutamate kinase ProB; (iii) Producing amino acids, wherein the amino acids are proline and trans-4-hydroxy-L-proline.

9. The use according to claim 8, characterized in that, The glutamate kinase ProB is a glutamate kinase that has been relieved of L-proline feedback inhibition.

10. The use according to claim 9, characterized in that, The glutamate kinase ProB is defined as the D107A amino acid mutation introduced into the encoding gene of glutamate kinase ProB (GenBank ID NP_414777.1) that can relieve L-proline feedback inhibition.

11. A method for enhancing the expression of a target gene, characterized in that, The method includes the step of operatively linking the polynucleotide with expression regulatory activity as described in claim 1 to the target gene.

12. A method for preparing proteins, characterized in that, The protein is expressed by the polynucleotide with expression regulatory activity according to claim 1, the transcription expression cassette according to claim 2 or 3, the recombinant expression vector according to claim 4, or the recombinant host cell according to any one of claims 5-7, wherein the protein is glutamate kinase ProB.

13. The method according to claim 12, characterized in that, The glutamate kinase ProB is a glutamate kinase that has been relieved of L-proline feedback inhibition.

14. The method according to claim 13, characterized in that, The glutamate kinase ProB is defined as the D107A amino acid mutation introduced into the encoding gene of glutamate kinase ProB (GenBank ID NP_414777.1) that can relieve L-proline feedback inhibition.

15. A method for producing amino acids, characterized in that, Choose the polynucleotide with expression regulatory activity according to claim 1, the transcription expression cassette according to claim 2 or 3, the recombinant expression vector according to claim 4, or the recombinant host cell expression amino acid synthesis-related enzyme according to any one of claims 5-7, and produce the amino acid in the presence of the amino acid synthesis-related enzyme, wherein the amino acid is proline or trans-4-hydroxy-L-proline, and the amino acid synthesis-related enzyme is glutamate kinase ProB.

16. The method according to claim 15, characterized in that, The glutamate kinase ProB is a glutamate kinase that has been relieved of L-proline feedback inhibition.

17. The method according to claim 16, characterized in that, The glutamate kinase ProB is defined as the D107A amino acid mutation introduced into the encoding gene of glutamate kinase ProB (GenBank ID NP_414777.1) that can relieve L-proline feedback inhibition.

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