Patent Application
20230279366
The present disclosure belongs to the field of biotechnology, and specifically relates to mutants of glutamate dehydrogenase gene promoters and applications thereof.
Amino acids, including glutamic acid, lysine, proline, etc., are basic substances constituting the proteins necessary for animal nutrition, and are widely used in industries such as medicine, health, food, animal fodder, and cosmetics. Amino acids are mainly produced by microbial fermentation. At present, the main production strains include microorganisms such as the genus Enterobacter and the genus Corynebacterium. Due to the physiological superiority of Corynebacterium, it has become the most important production strain in the industry. With the continuous development of the biotechnology, reports on metabolic engineering of Corynebacterium to improve its amino acid yield are gradually raising. These engineering modifications include enhancement of the expression of enzymes associated with synthetic pathways of the amino acids, weakening of the expression of enzymes associated with competition pathways, and so forth.
Glutamate dehydrogenase (encoded by the gdh gene) may catalyze the α-ketoglutaric acid to generate glutamic acid and is a key enzyme for biosynthesis of glutamic acid, and meanwhile glutamic acid is a synthesis precursor or important metabolite of proline, lysine, or the like. It has been reported in literatures that enhanced expression of key enzymes (such as glutamate dehydrogenase) in the synthetic pathways of target products can increase the yields of glutamic acid, lysine, proline, arginine, and the like in strains [1-4]. It is known to those skilled in the art that there are many ways to enhance the expression of enzymes, e.g., an increase in the copy number and replacement with a strong promoter. Increasing the copy number will increase the burden of strains to some extent and may lead to genomic instability of the strains. However, the regulation of gene expression by promoters does not have the above deficiencies. Therefore, from the standpoint of improving the productivity of amino acids of Corynebacterium, there is a need in the art to develop abundant promoter elements to appropriately enhance the expression of target genes such as gdh, thereby improving the productivity of amino acids of strains and derivatives thereof, etc.
In a first aspect, the present disclosure provides a mutant of a glutamate dehydrogenase gene promoter, wherein the mutant (i) has mutated nucleotide(s) at one or more positions corresponding to positions 479 to 482 of the nucleotide sequence set forth in SEQ ID NO: 30, and has mutated nucleotide(s) at one or more positions corresponding to positions 499 to 501 of the nucleotide sequence set forth in SEQ ID NO: 30.
The term “mutant” used herein refers to a polynucleotide or polypeptide that, relative to the “wild-type” or “comparative” polynucleotide or polypeptide, contains alternation(s) (i.e., substitution, insertion and/or deletion of a polynucleotide) at one or more (e.g., several) positions, where the substitution refers to a substitution of a different nucleotide for a nucleotide that occupies one position; the deletion refers to removal of a nucleotide that occupies certain position; and the insertion refers to an addition of a nucleotide after the nucleotide adjacent to and immediately following the occupied position.
Specifically, the mutant of the glutamate dehydrogenase gene promoter of Corynebacterium glutamicum has mutated nucleotide(s) at position(s) corresponding to one, two, three, or four positions in the nucleotide sequence corresponding to positions 479 to 482 of the nucleotide sequence set forth in SEQ ID NO: 30, and has mutated nucleotide(s) at position(s) corresponding to one, two, or three positions in the nucleotide sequence corresponding to positions 499 to 501 of the nucleotide sequence set forth in SEQ ID NO: 30. Moreover, the mutant of the glutamate dehydrogenase gene promoter of Corynebacterium glutamicum has an enhanced promoter activity as compared to the glutamate dehydrogenase gene promoter of Corynebacterium glutamicum.
In some embodiments, the mutant comprises a substituted nucleotide. The substitution is a mutation caused by a substitution of a base in the nucleotide with another different base, which is also referred to as a base substitution or a point mutation.
In some embodiments, the mutant (ii) comprises a reverse complementary sequence to the nucleotide sequence set forth in (i).
In some embodiments, the mutant (iii) comprises a reverse complementary sequence to a sequence capable of hybridizing with the nucleotide sequence set forth in (i) or (ii) under high-stringent hybridization conditions or very high-stringent hybridization conditions.
In some embodiments, the mutant (iv) comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in (i) or (ii). Specifically, the mutant comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence set forth in (i) or (ii).
Furthermore, the nucleotide sequence of the mutant set forth in any one of (i) to (iv) is not aattctttgtggtcatatctgtgcgacactgccataattgaacgtg at positions corresponding to position 469 to position 514 of the sequence set forth in SEQ ID NO: 30; and the mutant set forth in any one of (i) to (iv) has an enhanced promoter activity as compared to the glutamate dehydrogenase gene promoter having the sequence set forth in SEQ ID NO: 30.
In some embodiments, the nucleotide sequence of the mutant of the glutamate dehydrogenase gene promoter according to the present disclosure is mutated at position(s) corresponding to positions 479 to 482 of SEQ ID NO: 30 and mutated at position(s) corresponding to positions 499 to 501 of SEQ ID NO: 30.
Furthermore, the mutation occurring at position(s) corresponding to positions 479 to 482 of SEQ ID NO: 30 refers to the mutation of one or more bases corresponding to position 479, 480, 481, or 482 of SEQ ID NO: 30, and the mutation occurring at position(s) corresponding to positions 499 to 501 of SEQ ID NO: 30 refers to the mutation of one or more bases corresponding to position 499, 500, or 501 of SEQ ID NO: 30.
In some embodiments, the mutant of the glutamate dehydrogenase gene promoter according to the present disclosure has an enhanced promoter activity of 2 to 47 folds or more as compared to the glutamate dehydrogenase gene promoter having the sequence set forth in SEQ ID NO: 30.
In some embodiments, the nucleotide sequence of the promoter core region of the mutant of the glutamate dehydrogenase gene promoter according to the present disclosure is one of the following sequences:
In some embodiments, the nucleotide sequence of the mutant of the glutamate dehydrogenase gene promoter according to the present disclosure is a sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 29.
The mutant of the glutamate dehydrogenase gene promoter is a nucleotide molecule having a promoter activity. The “promoter” refers to a nucleic acid molecule, which is typically located upstream of the coding sequence of a gene of interest, providing a recognition site for the RNA polymerase, and is located upstream of the 5′ direction of the start site of mRNA transcription. It is the untranslated nucleic acid sequence to which the RNA polymerase binds to initiate transcription of the gene of interest.
The “promoter core region” refers to a nucleic acid sequence located on the promoter in a prokaryote, which is the core sequence region where the function of the promoter is exerted, mainly including the −35 region, the −10 region, the region between the −35 region and the −10 region, and the transcription starting site, the −35 region is a recognition site of the RNA polymerase, the −10 region is a binding site of the RNA polymerase. The mutant of the promoter of the present disclosure is an improved promoter of the gdh gene of Corynebacterium glutamicum, which has a higher promoter activity than that of the wild-type promoter, e.g., increasing at least by 2.3 folds or more, preferably 3.3 folds or more, 5 folds or more, 8 folds or more, more preferably 10 folds or more, 11 folds or more, 12 folds or more, 13 folds or more, 14 folds or more, and most preferably 40 folds or more, 45 folds or more.
The nucleic acid molecule of the promoter of the present disclosure may be isolated or prepared by a standard molecular biology technology. For example, the nucleic acid molecule of the promoter of the present disclosure may be isolated by PCR using suitable primer sequences. Also, the nucleic acid molecule of the promoter of the present disclosure may be prepared by a standard synthetic technique using an automated DNA synthesizer.
In a second aspect, the present disclosure provides an expression cassette and a recombinant vector comprising the mutant of the glutamate dehydrogenase gene promoter.
The “expression cassette” has the meaning generally understood by a person skilled in the art, i.e., an element containing a promoter and a gene of interest and is capable of expressing the gene of interest.
The term “vector” refers to a DNA construct containing DNA sequences operably ligated to appropriate regulatory sequences, so as to express a gene of interest in a suitable host. The vector used herein is not particularly limited, and may be any vector known in the art as long as it is capable of replicating in a host. That is, the vector includes, but is not limited to, a plasmid and a phage, for example, the pEC-XK99E plasmid used in the specific examples of the present disclosure. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or integrate into the genome per se in some cases.
In a third aspect, the present disclosure provides a recombinant host cell comprising the mutant of the glutamate dehydrogenase gene promoter, the expression cassette, or the recombinant vector.
The recombinant host cell is specifically achieved by transformation. The term “transformation” used herein has the meaning generally understood by a person skilled in the art, namely, a process of introducing an exogenous DNA into a host. Methods for the transformation include any method for introducing a nucleic acid into a cell. These methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2)) precipitation, microinjection, a polyethylene glycol (PEG) method, a DEAE-dextran method, a cationic liposome method, and a lithium acetate-DMSO method.
The “host cell” used herein has the meaning generally understood by a person skilled in the art, i.e., the cell that can be introduced with the nucleic acid having the promoter activity according to the present disclosure, and is referred to as a recombinant host cell after introduction. In other words, any host cell may be used in the present disclosure as long as it contains the nucleic acid having the promoter activity according to the present disclosure and is operably ligated to a gene to mediate transcription of this gene. The host cell of the present disclosure may be a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell of the present disclosure may be any type of strains capable of producing the target product, which include wild-type strains and recombinant strains. Exemplarily, the host cell is derived from microorganisms suitable for fermentation production of target products such as amino acids and organic acids, e.g. Enterobacterium, Corynebacterium, Brevibacterium, Arthrobacterium, and Microbacterium etc.
In some preferred embodiments, the host cell is Enterobacterium or Corynebacterium, more preferably Corynebacterium glutamicum, including, but not limited to, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum B253, Corynebacterium glutamicum ATCC 14067, and L-amino acids-producing derived strains prepared from the strains described above.
In a specific embodiment of the present disclosure, the host cell is Corynebacterium glutamicum, and the Corynebacterium glutamicum is further modified, specifically by introducing a G149K mutation coding sequence into the coding gene of glutamate kinase ProB in the bacterium, so as to relieve proline feedback inhibition. In some more specific embodiments, in proline feedback inhibition relieved strains, the expression cassette overexpressing glutamate kinase proBG149K, glutamate-5-semialdehyde dehydrogenase proA, and pyrroline-5-carboxylate dehydrogenase proC using a pyc promoter is further integrated into the proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase putA gene to obtain proline-producing strains. Preferably, the pyc promoter is an enhanced promoter by mutation; further preferably, the sequence of the core region of the promoter is set forth in SEQ ID NO: 68; and more preferably, the sequence of the pyc promoter is set forth in SEQ ID NO: 69. In this way, proline can be generated more efficiently.
In other embodiments, the host cell may also be other types of amino acids-producing strains. The “amino acids-producing strain” described herein refers to the strain that can produce amino acids when bacteria are cultured in a medium and could accumulate amino acids, or can secrete amino acids into the medium, i.e., extracellular free amino acids are obtainable. For example, the strains may be naturally occurring amino acids-producing strains, or engineered amino acids-producing strains obtained by genetic modifications.
Exemplarily, the host cell is a lysine-producing host cell. In some embodiments, the lysine-producing host cells may be strains expressing the aspartate kinase that relieves the feedback inhibition based on Corynebacterium glutamicum ATCC 13032. In addition, the lysine-producing host cells may also be derived strains having the lysine-producing capability.
In some embodiments, the lysine-producing host cells may further include, but not limited to, one or more genes selected from one or more of the following that are attenuated or reduced in expression:
i. thrB gene encoding homoserine kinase;
j. ldcC gene encoding lysine decarboxylase; and
h. cadA gene encoding lysine decarboxylase.
In some embodiments, the lysine-producing host cells may further include, but not limited to, one or more genes selected from one or more of the following that are enhanced or overexpressed:
Exemplarily, the host cells are threonine-producing host cells. In some embodiments, the threonine-producing host cells may be strains expressing the aspartate kinase LysC that relieves the feedback inhibition based on Corynebacterium glutamicum ATCC 13032. In other embodiments, the threonine-producing host cells may also be other types of strains having the threonine-producing capability.
In some embodiments, one or more genes selected from the following are enhanced or overexpressed in the threonine-producing host cells:
Exemplarily, the host cells are isoleucine-producing host cells. In some embodiments, the isoleucine-producing host cells are strains that produce L-isoleucine by substituting the amino acid at position 323 of the threonine dehydratase encoded by the ilvA gene with alanine. In other embodiments, the isoleucine-producing host cells may also be other types of strains having the isoleucine-producing capability.
Exemplarily, the host cells are O-acetylhomoserine-producing host cells. In some embodiments, the O-acetylhomoserine-producing host cells are strains that produce O-acetylhomoserine by inactivating O-acetylhomoserine(thiol)-lyase. In other embodiments, the O-acetylhomoserine-producing host cells may also be other types of strains having the O-acetylhomoserine-producing capability.
Exemplarily, the host cells are methionine-producing host cells. In some embodiments, the methionine-producing host cells are strains that produce methionine by inactivating the transcriptional regulators of methionine and cysteine. In other embodiments, the methionine-producing host cells may also be other types of strains having the methionine-producing capability.
In the present disclosure, the host cells may be cultured by conventional methods in the art, including, but not limited to, well-plate culture, shaking culture, batch culture, continuous culture, fed-batch culture, etc. Also, various culture conditions such as the temperature, time, and pH value of the medium may be properly adjusted according to actual situations.
In a fourth aspect, the present disclosure provides use of the mutant of the glutamate dehydrogenase gene promoter according to the first aspect, the expression cassette or expression vector according to the second aspect, or the recombinant host cell according to the third aspect in at least one of the following (a) to (c):
In some embodiments, the protein is a target product synthesis-associated protein, a membrane transport-associated protein, or a gene expression regulatory protein.
In some embodiments, the target product may be selected from at least one of amino acids and derivatives thereof, or may be selected from other kinds of compounds that may be biosynthetically available in the art.
Exemplarily, the amino acids and derivatives thereof include, but are not limited to, one or a combination of two or more of the following: proline, hydroxyproline, lysine, glutamic acid, arginine, ornithine, glutamine, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, methionine, aspartic acid, asparagine, histidine, phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid, or derivatives of any one of the derivatives described above.
In a fifth aspect, the present disclosure provides a method for enhancing expression of a target gene, the method comprising operably ligating the mutant of the glutamate dehydrogenase gene promoter to a target gene.
The term “operably ligated” used herein means that the mutant of the glutamate dehydrogenase gene promoter of the present disclosure is functionally ligated to the coding gene to initiate and mediate transcription of the gene, indicating that the mutant of the glutamate dehydrogenase gene promoter of the present disclosure is operably ligated to the coding gene to control the transcriptional activity of the operon gene. The methods of the operable ligating may be any method described in the art. In case of using the mutant of the glutamate dehydrogenase gene promoter according to the present disclosure, the method of enhancing expression of a target gene according to the present disclosure includes the methods commonly adopted by a person skilled in the art.
Exemplarily, the coding gene includes, but is not limited to, a coding gene of a target product synthesis-associated protein, a coding gene of a membrane transport-associated protein, or a coding gene of a gene expression regulatory protein.
In some embodiments, the mutant of the glutamate dehydrogenase gene promoter is used for expression of the gene associated with amino acid synthesis. Exemplarily, the mutant of the glutamate dehydrogenase gene promoter may be used for expression of the other genes in Corynebacterium or Enterobacterium, including, but not limited to, genes associated with amino acid synthesis, e.g., glutamate dehydrogenase Gdh, aspartate kinase LysC, threonine operon ThrABC, aspartate-semialdehyde dehydrogenase Asd, aspartate-ammonia lyase AspB, homoserine dehydrogenase Hom, homoserine O-acetyltransferase MetX, dihydrodipicolinate synthase DapA, dihydrodipicolinate reductase DapB, meso-diaminopimelate dehydrogenase Ddh, glutamate kinase ProB, glutamate-5-semialdehyde dehydrogenase ProA, pyrroline-5-carboxylate dehydrogenase ProC, proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase PutA, glutamyl-t-RNA reductase hemA, pyruvate carboxylase Pyc, phosphoenolpyruvate carboxylase Ppc, an amino acid transport protein, a ptsG system-associated protein, pyruvate dehydrogenase AceE, glyceraldehyde-3-phosphate dehydrogenase GapN, or lysine decarboxylase CadA or LdcC.
In one specific embodiment, the coding gene is the gene of the glutamate dehydrogenase, which may catalyze the α-ketoglutaric acid to generate glutamic acid and is a key enzyme for biosynthesis of glutamic acid, and meanwhile glutamic acid is a synthesis precursor of proline, lysine, or the like. A large number of literatures have reported that enhanced expression of the glutamate dehydrogenase can increase the yields of glutamic acid, proline, lysine, arginine, and the like in strains [1-4]. The strength-based promoter elements of the present disclosure can be used to moderately regulate the expression of the target genes and achieve high-efficient production of the target products.
Further, in a sixth aspect, the present disclosure provides a method for preparing a protein, wherein the method comprises a step of expressing the protein using the expression cassette or expression vector according to the second aspect, or the recombinant host cell according to the third aspect; optionally, the protein is a target product synthesis-associated protein, a membrane transport-associated protein, or a gene expression regulatory protein; and
Optionally, the method further comprises a step of isolating or purifying the protein.
Further, in a seventh aspect, the present disclosure provides a method for producing a target product, the method comprising culturing a host cell containing the mutant of the glutamate dehydrogenase gene promoter according to the first aspect that is operably ligated to a gene associated with synthesis of the target product, and collecting the resulting target product.
In some embodiments, the target product is an amino acid. The method for producing the amino acid comprises culturing a host cell containing the mutant of the glutamate dehydrogenase gene promoter that is operably ligated to the gene encoding the glutamate dehydrogenase, and collecting the produced amino acids. Wherein, the amino acid includes proline, lysine, glutamic acid, hydroxyproline, arginine, ornithine, glutamine, etc. More preferably, the gene associated with production of the amino acid includes, but is not limited to, the gdh gene, the lysC gene, the thrABC gene, the asd gene, the aspB gene, the horn gene, the metX gene, the dapA gene, the dapB gene, the ddh gene, the proB gene, the proA gene, the proC gene, the putA gene, the hemA gene, the pyc gene, the ppc gene, the lysE gene, the ptsG gene, the ptsI gene, the aceE gene, the gapN gene, etc. With the methods disclosed herein, it is possible to improve the yield of amino acids of strains, in particular proline, lysine, glutamic acid, hydroxyproline, arginine, ornithine, glutamine, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, methionine, aspartic acid, asparagine, histidine, phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid, or derivatives of any one of the amino acids described above.
The method for producing amino acids according to the present disclosure is the method commonly used by a person skilled in the art on the basis of using the mutant of the glutamate dehydrogenase gene promoter of the present disclosure, and meanwhile the method comprises a step of recovering an amino acid from a cell or a culture solution. The method for recovering amino acids from the cell or medium is well-known in the art, and includes, but is not limited to, filtration, anion exchange chromatography, crystallization, and HPLC.
The present disclosure achieves the following advantageous effects: the nucleic acid molecule with an enhanced promoter activity provided herein exhibits a higher promoter activity than that of a wild type, and is useful for enhancing expression of the gene of interest. For example, when operably linked to the gdh gene, etc., the nucleic acid molecule can enhance the expression intensity of target product synthesis-associated proteins such as the glutamate dehydrogenase, thereby improving the production efficiency of amino acids and organic acids of the recombinant strains, so it has a relatively high application value.
When used in combination with the term “including” in the claims and/or specification, the word “a” or “an” may refer to “one”, or refer to “one or more”, “at least one”, and “one or more than one”.
As used in the claims and specification, the term “including”, “having”, “comprising”, or “containing” is intended to be inclusive or open-ended, and does not exclude additional or unrecited elements or methods and steps.
Throughout the application document, the term “about” means that a value includes the error or standard deviation caused by the device or method used to measure the value.
It is applicable to the content disclosed herein that the term “or” is defined only as alternatives and “and/or”, but the term “or” used herein refers to “and/or” unless otherwise expressly stated to be only alternatives or mutual exclusion between alternatives.
The selected/optional/preferred “numerical range”, when used in the claims or the specification, includes not only the numerical endpoints at both ends of the range, but also all natural numbers covered between the above numerical endpoints with respect to these numerical endpoints.
As used herein, the term “nucleic acid molecule” or “polynucleotide” refers to a polymer composed of nucleotides. A nucleic acid molecule or polynucleotide may be in the form of an individual fragment, or may be a constituent part of a larger nucleotide sequence structure, which is derived from the nucleotide sequence that has been isolated at least once in number or concentration, and could be recognized, operated, and sequence recovered as well as nucleotide sequence recovered by a standard molecular biological method (e.g., using a cloning vector). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes an RNA sequence (i.e., A, U, G, C), where “U” substitutes for “T”. In other words, “polynucleotide” refers to a nucleotide polymer knocked out from an additional nucleotide (an individual fragment or an entire fragment), or may be a constituent part or component of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.
As used herein, the terms “target gene” and “gene of interest” can be used interchangeably.
As used herein, the term “wild-type” refers to an object that can be found in nature. For example, a polypeptide or a polynucleotide sequence present in an organism that can be isolated from one source in nature and has not been intentionally modified by human in the laboratory is naturally occurring. As used herein, “naturally occurring” and “wild-type” are synonymous.
As used herein, the terms “sequence identity” and “percent identity” refer to the percentage of nucleotides or amino acids that are the same (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides may be determined by aligning the nucleotide sequences of polynucleotides or the amino acid sequences of polypeptides and scoring the number of positions at which nucleotide or amino acid residues are identical in the aligned polynucleotides or polypeptides, and comparing the number of these positions with the number of positions at which nucleotide or amino acid residues are different in the aligned polynucleotides or polypeptides. Polynucleotides may differ at one position by, e.g., containing a different nucleotide (i.e., substitution or mutation) or deleting a nucleotide (i.e., insertion or deletion of a nucleotide in one or two polynucleotides). Polypeptides may differ at one position by, e.g., containing a different amino acid (i.e., substitution or mutation) or deleting an amino acid (i.e., insertion or deletion of an amino acid in one or two polypeptides). The sequence identity may be calculated by dividing the number of positions at which nucleotide or amino acid residues are identical by the total number of nucleotide or amino acid residues in the polynucleotides or polypeptides. For example, the percent identity may be calculated by dividing the number of positions at which nucleotide or amino acid residues are identical by the total number of nucleotide or amino acid residues in the polynucleotides or polypeptides, and multiplying the result by 100.
In some embodiments, two or more sequences or subsequences, when compared and aligned at maximum correspondence by the sequence alignment algorithm or by the visual inspection measurement, have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% “sequence identity” or “percent identity” of nucleotides. In some embodiments, the sequence is substantially identical over the full length of either or both of the compared biopolymers (e.g., polynucleotides).
As used herein, the term “complementary” refers to hybridization or base pairing between nucleotides or nucleotides, e.g., between two chains of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleotide to be sequenced or amplified.
As used herein, the term “high-stringent conditions” means that following the standard DNA blotting procedures, a probe of at least 100 nucleotides in length pre-hybridizes or hybridizes for 12 to 24 hours at 42° C. in 5×SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg/ml of cleavaged and denatured salmon sperm DNA, and 50% formamide. Finally, the vector material is washed three times at 65° C. with 2×SSC and 0.2% SDS, each for 15 min.
As used herein, the term “very high-stringent conditions” means that following the standard DNA blotting procedures, a probe of at least 100 nucleotides in length pre-hybridizes or hybridizes for 12 to 24 hours at 42° C. in 5×SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg/ml of cleavage and denatured salmon sperm DNA, and 50% formamide. Finally, the vector material is washed three times at 70° C. with 2×SSC and 0.2% SDS, each for 15 min.
Unless otherwise defined, all technical and scientific terms used herein have the same meanings as typically understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein may be used to implement or test the present disclosure, the methods and materials described herein are preferred.
The present disclosure will be further illustrated with reference to specific examples. It should be appreciated that these examples are merely intended to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental techniques and experimental methods used in the examples, unless otherwise specified, are all conventional techniques and methods, for example, experimental methods for which no specific conditions are indicated in the following examples are generally performed according to conventional conditions such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by manufacturers. The materials, reagents, etc. used in the examples, unless otherwise specified, are all commercially available.
In order to characterize the strength of the gdh gene promoter of Corynebacterium glutamicum, the present disclosure firstly constructed a characterizing vector, and based on the pEC-XK99E plasmid skeleton, 60 amino acids at the N-terminus of the gdh gene, a C-peptide, and a red fluorescence protein gene were expressed by the gdh gene promoter. Based on the disclosed genome sequence and gdh gene annotation information of Corynebacterium glutamicum ATCC13032, a primer gdh-FIR was designed, and a 180-bp DNA fragment of the gdh gene promoter along with the N-terminus was obtained by PCR amplification using the ATCC13032 genome as a template. The pEC-XK99E-rfp[5] plasmid reported in the literature was used as a template and pEC-F/R as a primer to amplify the DNA fragment of the pEC-XK99E plasmid skeleton, the C-peptide, along with the red fluorescence protein gene. The above two fragments were ligated via Vazyme's One Step Cloning Kit to obtain a pEC-XK99E-Pgdh-rfp characterizing vector, and its plasmid vector map was shown in
(1) Construction of Library of Gdh Gene Promoter Mutants of Corynebacterium glutamicum
The present disclosure performed mutations on the core region of the gdh gene promoter of Corynebacterium glutamicum “AATTCTTTGTGGTCATATCTGTGCGACACTGCCATAATTGAACGTG”, where the underlined parts were the main sequences of the −35 region and the −10 region of the promoter. The present disclosure performed mutations on the corresponding positions of the above core region, obtaining “AATTCTTTGTNNNNATATCTGTGCGACACTNNNATAATTGAACGTG”. The two fragments of the plasmid were amplified using gdh-M1/M2 and gdh-M3/M4 primers respectively, and ligated via Vazyme's One Step Cloning Kit. All of the resulting cloned bacteria were collected and plasmids were extracted to obtain a library of the gdh gene promoter mutants. The above library and the wild-type control pEC-XK99E-Pgdh-rfp obtained in Example 1 were transformed into Corynebacterium glutamicum ATCC13032 respectively, and coated on a TSB plate. The plate, on which hundreds of clones were grown, was fluorescence photographed with a fluorescence imaging system. Mutants with improved expression intensity were preliminarily screened according to the fluorescence brightness of the clones. The ingredients (g/L) of the medium of the TSB plate were as follows: glucose, 5 g/L; yeast powder, 5 g/L; soy peptone, 9 g/L; urea, 3 g/L; succinic acid, 0.5 g/L; K2HPO4·3H2O, 1 g/L; MgSO4·7H2O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; MOPS, 20 g/L; and agar powder, 15 g/L. The present disclosure performed initial screening on more than 10,000 clones, and about 40 mutants with enhanced fluorescence intensity were obtained (the screening plate was as shown in
(2) Strength Characterization of Library of Gdh Gene Promoter Mutants of Corynebacterium glutamicum
All mutants with the enhanced fluorescence intensity observed in the plate as described above were cultured in a 96-well plate to characterize the strength of the promoters. The ingredients (g/L) of the TSB liquid medium were as follows: glucose, 5 g/L; yeast powder, 5 g/L; soy peptone, 9 g/L; urea, 3 g/L; succinic acid, 0.5 g/L; K2HPO4·3H2O, 1 g/L; MgSO4·7H2O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; and MOPS, 20 g/L. The strains resulting from the plate were inoculated with toothpicks into a 96-well plate containing 200 μl of TSB liquid medium in each well. Three samples were set for each strain. The rotating speed of the plate shaker was 800 rpm. After culturing at 30° C. for 24 h, the fluorescence intensities of the strains were measured, and the strains with improved fluorescence intensity as compared to the wild-type control were sequenced. The results were listed in Table 3. Some of the promoter mutants have the same sequence. Finally, the present disclosure successfully obtained 29 different promoter mutants with improved expression intensity as compared to the wild-type promoter, and the range of the increased folds was from 2.3 folds to 46.4 folds. The promoter mutants were sequentially numbered as Pgdh-1 to Pgdh-29, which could provide abundant elements for modifying the expression of genes such as gdh.
(1) Evaluation of Construction of Proline-Producing Base Strain SLCgP2
It had been reported in literatures that the introduction of a G149K mutation in the glutamate kinase ProB could relieve the proline feedback inhibition, and introduction of this mutation in the genome of the Corynebacterium glutamicum ATCC13032 strain enabled production of proline. In the present disclosure, the G149K mutation was introduced in the Corynebacterium glutamicum ATCC13032 strain, and the codon was mutated from GGT to AAG, thereby obtaining a SLCgP1 strain.
The SLCgP1 strain was further applied in the present disclosure to integrate the expression cassette, which was over-expressed using the Ppyc-20 promoter (the nucleotide sequence of the core region was CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG as set forth in SEQ ID NO: 68, and the nucleotide sequence of the promoter was set forth in SEQ ID NO: 69), of the glutamate kinase proBG149K which relieves the feedback inhibition, glutamate-5-semialdehyde dehydrogenase proA along with pyrroline-5-carboxylate dehydrogenase proC into the proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase putA gene, to obtain a proline-producing strain designated as a SLCgP2 strain.
The SLCgP2 strain was specifically constructed as follows: 1) Firstly, constructing the expression cassette of proBG149K, proA, and proC along with Ppyc-20 promoter on the pEC-XK99E plasmid. The Ppyc-20 promoter fragment was amplified using a plasmid containing the Ppyc-20 promoter as a template and pyc-a/b as primers; the fragments of proBG149K, proA, and proC were amplified using the genome of the SLCgP1 strain as a template and proB-112, proA-112, and proC-1/2 as primers, respectively; and at the same time, the pEC-XK99E skeleton was amplified using pEC-1/2 as primers. The above 5 fragments were ligated via Vazyme's One Step Cloning Kit to obtain a pEC-proBG149KproAproC plasmid. 2) Constructing a recombinant vector that inserts the above expression cassette into the putA gene on the chromosome. The upstream and downstream homologous arms inserted into the putA gene were amplified using the genome of the SCgL30-Ppyc-20 strain as a template and putA-1/2 and putA-3/4 as primers, respectively; the expression cassette fragment was amplified using the pEC-proBG149KproAproC plasmid as a template and ABC-FIR as primers; and at the same time, the pK18mobsacB skeleton was amplified using pK18-1/2 as primers. The above PCR fragments were ligated via Vazyme's One Step Cloning Kit to obtain a pK18-proBG149KproAproC recombinant vector. 3) The pK18-proBG149KproAproC recombinant vector was transformed into the SLCgP1 strain. The strain was coated on an LBHIS solid medium containing 5 g/L of glucose and 25 μg/mL of kanamycin and cultured at 30° C. to obtain the first recombinant transformants. The correct first recombinant transformants were inoculated into LB media containing 5 g/L of glucose respectively, and cultured overnight. Thereafter, 1% of the culture was transferred into an LB medium containing 100 g/L of sucrose and cultured overnight at 30° C., and then coated respectively on an LB solid medium containing 100 g/L of sucrose for screening. Correct mutants were confirmed by PCR with universal and specific primers and sequencing to obtain SLCgP2 strains, respectively. The sequences of the primers as used above were listed in Table 4.
(2) Construction of Recombinant Vector of Gdh Gene Promoter Mutant of Corynebacterium glutamicum
Based on the reported genome sequence of Corynebacterium glutamicum ATCC13032, the upstream homologous arm was amplified using ATCC13032 genome as a template and gdh-UF/gdh-UR as primers respectively, and the downstream homologous arms of the Pgdh-1, Pgdh-7, Pgdh-16, Pgdh-23, Pgdh-26, and Pgdh-29 promoter mutants were amplified using gdh-DF1/gdh-DR, gdh-DF7/gdh-DR, gdh-DF16/gdh-DR, gdh-DF23 gdh-DR, gdh-DF26/gdh-DR, and gdh-DF29/gdh-DR as primers, respectively; and at the same time, the pK18mobsacB skeleton was amplified using pK18-1/2 as primers. The above PCR fragments were recovered and then ligated via Vazyme's One Step Cloning Kit, and recombinant vectors pK18-Pgdh-1, pK18-Pgdh-7, pK18-Pgdh-16, pK18-Pgdh-23, pK18-Pgdh-26, and pK18-Pgdh-29 with mutated promoters were obtained, respectively. The sequences of the primers as used above were listed in Table 5.
(3) Construction of Gdh Gene Promoter Mutants in Proline-Producing Strain of Corynebacterium glutamicum
The recombinant vectors pK18-Pgdh-1, pK18-Pgdh-7, pK18-Pgdh-16, pK18-Pgdh-23, pK18-Pgdh-26, and pK18-Pgdh-29 as constructed above were transformed into the proline-producing strains SLCgP2 of Corynebacterium glutamicum, respectively. The strains were coated on LBHIS solid media containing 5 g/L of glucose and 25 μg/mL of kanamycin and cultured at 30° C. to obtain the first recombinant transformants. The correct first recombinant transformants were inoculated into LB media containing 5 g/L of glucose respectively, and cultured overnight. Thereafter, 1% of the culture was transferred into an LB medium containing 100 g/L of sucrose and cultured overnight at 30° C., and then coated respectively on LB solid media containing 100 g/L of sucrose for screening. Correct mutants were confirmed by PCR and sequencing to obtain strains SLCgP3, SLCgP4, SLCgP5, SLCgP6, SLCgP7, and SLCgP8 with the mutated gdh promoters, respectively.
(4) Evaluation of Proline Productivity of Gdh Gene Promoter Mutants in Proline-Producing Strain of Corynebacterium glutamicum
In order to test how the mutation of the gdh promoter of Corynebacterium glutamicum affected production of proline by strains, fermentation tests were performed on SLCgP2, SLCgP3, SLCgP4, SLCgP5, SLCgP6, SLCgP7, and SLCgP8, respectively. The ingredients of the fermentation medium were as follows: glucose, 72 g/L; yeast powder, 1 g/L; soy peptone, 1 g/L; NaCl, 1 g/L; ammonium sulfate, 1 g/L; urea, 10 g/L; K2HPO4·3H2O, 1 g/L; MgSO4·7H2O, 0.45 g/L; FeSO4·7H2O, 0.05 g/L; biotin, 0.4 mg/L; vitamin B1, 0.1 mg/L; MOPS, 40 g/L; and initial pH7.2. The strains were firstly inoculated into TSB liquid media and cultured for 8 h. The cultures were inoculated as seeds into a 24-well plate containing 800 μl of fermentation media in each well at an inoculation amount of 12 and cultured at 30° C. for 18 h. The rotating speed of the plate shaker was 800 rpm. Three samples were set for each strain. After the fermentation, the proline yields were measured. The results were listed in Table 6. The proline yields of the strains after mutation of the gdh promoter were all significantly increased.
The above results suggest that mutants with enhanced expression intensity of the gdh gene promoter are applicable to the production of target products dependent upon the reaction from α-ketoglutaric acid to glutamic acid, for example, biological production of amino acids with glutamic acid as a product or a major synthesis precursor, such as proline, lysine, glutamic acid, hydroxyproline, arginine, ornithine, and glutamine. Asakura Y et al. have reported that the yield of glutamic acid could be increased by enhancing the Gdh activity using the gdh promoter mutant [6]. Xu, J et al. have reported that the lysine yield could be increased by enhancing the Gdh activity through substitution of the Ptac-M promoter for the promoter of the gdh gene [7]. Hydroxyproline can be produced by one-step hydroxylation using proline as a precursor. The modification for improving the synthesis of proline described herein is also useful for production of hydroxyproline.
All technical features disclosed in the present specification may be combined in any combination manner. Each of the technical features disclosed in the present specification may be substituted by other features having the same, equivalent or similar effects. Thus, unless specifically stated, each of the features disclosed herein is only an example of a series of equivalent or similar features.
In addition, with the foregoing descriptions of the present disclosure, the key features of the present disclosure become apparent to a person skilled in the art. Many modifications may be made to the invention without departing from the spirit and scope of the present disclosure, so as to adapt the invention to various application purposes and conditions. Therefore, such modifications are also intended to fall within the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010696984.7 | Jul 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/105953 | 7/13/2021 | WO |
