CN116891868A - Osmotic pressure tolerance functional gene identification method and application of functional gene - Google Patents

Osmotic pressure tolerance functional gene identification method and application of functional gene Download PDF

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CN116891868A
CN116891868A CN202310256561.7A CN202310256561A CN116891868A CN 116891868 A CN116891868 A CN 116891868A CN 202310256561 A CN202310256561 A CN 202310256561A CN 116891868 A CN116891868 A CN 116891868A
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amino acid
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protein
corynebacterium glutamicum
osmotic pressure
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孙际宾
王钰
赵晓佳
郑平
刘娇
王猛
周文娟
陈久洲
郭轩
程海娇
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The application discloses an osmotic pressure tolerance functional gene identification method and application of a functional gene. The application firstly constructs a functional gene screening method based on a whole genome single gene overexpression library of corynebacterium glutamicum, which is used for rapidly screening functional genes capable of improving the tolerance of strains to high osmotic pressure in a high-throughput manner. The tolerance of the strain to high osmotic pressure is improved by over-expressing the functional genes, so that the amino acid yield of the strain is improved.

Description

Osmotic pressure tolerance functional gene identification method and application of functional gene
Cross Reference to Related Applications
The present application claims priority and benefit from the chinese application patent application No. 202210349424.3 filed on month 01 of 2022, incorporated herein by reference in its entirety.
Technical Field
The application belongs to the field of genetic engineering, and in particular relates to a method for identifying functional genes of osmotic pressure tolerance of microorganisms, a method for improving the tolerance of microorganisms to osmotic pressure by utilizing the functional genes obtained by screening, and a method for increasing the amino acid yield of strains.
Background
The corynebacterium has the advantages of biosafety (FDA certification in the United states), high growth speed, low nutrition demand, wide substrate spectrum and the like, is considered as an ideal industrial chassis strain in the biological fermentation industry, and is widely applied to the industrial production of chemicals such as amino acid, organic acid and the like, and particularly has a dominant role in the fermentation production of amino acid such as lysine, glutamic acid and the like. Numerous studies and genetic engineering of coryneform strains have been carried out in the prior art for many years, including engineering of amino acid synthesis pathways, substrate utilization pathways, enhanced amino acid excretion, etc., and higher levels of engineered coryneform strains producing amino acids have been obtained. Currently, the industrial fermentation level of various amino acids exceeds 100g/L, and the industrial fermentation level of lysine exceeds 200g/L (Xu, J.Z., et al, microb Cell face, 2020,19,39; hehongtong et al, chinese brewing, 2018,37 (10), 51-56).
However, in the fermentation process, accumulation of fermentation products such as high-concentration lysine, glutamic acid and the like and continuous addition of substrates all cause extremely high osmotic pressure on coryneform bacteria cells, and the high osmotic pressure is almost environmental stress (Varela C et al applied Microbiology And Biotechnology 2003,60 (5): 547-555) which all high-level industrial strains face in the later period of fermentation, directly influences the expansion degree, the integrity, the hydration degree, the molecular crowding degree and the like of the cells, thereby interfering the growth metabolism of the cells, becoming a key factor influencing the synthesis efficiency of the final products and limiting further improvement of the fermentation level of the industrial strains. The capability of the corynebacterium strain to withstand high osmotic pressure is enhanced, and the method is an effective means for improving the final concentration of fermentation products and improving the economy of biological fermentation. However, in the prior art, due to the lack of a high-throughput functional gene identification method, there are few osmotically tolerant functional genes derived from corynebacteria that have been currently mined, thereby limiting further improvement of the fermentation performance of corynebacteria.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention firstly constructs a high-throughput screening method of functional genes based on a whole genome single gene overexpression library of corynebacterium glutamicum, which is used for rapidly screening the functional genes capable of improving the tolerance of strains to high osmotic pressure in a high throughput manner, thereby improving the amino acid yield of the strains. The present invention has been completed on the basis of this.
The specific technical scheme of the invention is as follows:
in a first aspect, a high throughput identification method of a functional gene of osmotic pressure tolerance of a microorganism is provided, specifically comprising the following steps:
1) Constructing a single gene overexpression library based on a microbial whole genome, and constructing a control plasmid strain without cloning any gene sequence in the middle;
2) Preliminary screening of the over-expression growth improvement gene under the condition of high osmotic pressure of lysine;
3) And (3) re-screening the genes by over-expressing and growing the genes under the conditions of high osmotic pressure and non-high osmotic pressure of lysine.
Preferably, the microorganism is a microorganism of the genus corynebacterium; further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
Preferably, the step 2) is specifically to transfer the microorganism whole genome monogenic overexpression strain constructed in the step 1) and seed solution containing the control plasmid strain into lysine hypertonic culture medium respectively, and culture and OD determination are carried out by adopting a MicroScreen full-automatic microorganism growth curve analyzer 600 OD at each time point of all over-expressed strains 600 Calculating the ratio with the control plasmid strain, normalizing all batches of data, calculating the average value of 15 continuous maximum values in the growth process of the end point for 30 hours, and judging the genes with the 15 values improved by more than 20 percent as growth improvement genes.
Further preferably, the culture system of step 2) is a 48-well plate, 400. Mu.L of the loading liquid, the culture medium contains 0.04mM IPTG and 25. Mu.g/mL kana antibiotics, the culture is carried out at 30 ℃ and 800rpm for 96-106 hours, and the components of the hypertonic culture medium are (g/L): (NH) 4 ) 2 SO 4 ，20g/L；Uera，5g/L；KH 2 PO 4 ，1g/L；K 2 HPO 4 ·3H 2 O，1.3g/L；MOPS，42g/L；CaCl 2 ，0.01g/L；FeSO 4 ·7H 2 O，0.01g/L；MnSO 4 ·H 2 O，0.01g/L；ZnSO 4 ·7H 2 O，0.001g/L；CuSO 4 ，0.0002g/L；NiCl·6H 2 O，0.00002g/L；MgSO 4 ·7H 2 O,0.25g/L; protocatechuic acid, 0.03g/L; VB1,0.0001g/L; biotin, 0.0002g/L; yeast powder, 2g/L; glucose, 40g/L; lysine, 175g/L; h 2 SO 4 The pH is regulated to 7.1-7.2.
Preferably, the step 3) is specifically to over-express the growth-enhancing gene strain obtained by the preliminary screening in the step 2) and the strain containing the samePerforming re-screening of lysine with high osmotic pressure and non-high osmotic pressure by controlling the plasmid strain; cultivation and determination of OD Using MicroScreen fully automatic microorganism growth Curve Analyzer 600 3 parallel samples of all strains OD at each time point 600 Calculating average value and standard deviation respectively, and drawing a growth curve of each strain under non-hypertonic and hypertonic conditions; the strain overexpressed by the sample at each time point during the 30h end point growth process was analyzed again with the strain OD containing the control plasmid 600 Significance of difference (T-test analysis), P if hypertonic conditions are satisfied for 15 consecutive time points and above<0.05, and the overexpression strain OD of these spots 600 The average value is larger than that of the control strain, so that the over-expression of the gene can improve the strain growth under the condition of lysine hyperpermeability.
Further preferably, the culture system of step 3) is a 48-well plate containing 400. Mu.L of liquid, 0.04mM IPTG and 25. Mu.g/mL kana antibiotic are added, the culture is carried out at 30 ℃ for 96-106 hours at 800rpm, and OD is detected every 2 hours 600 Three experiments were set up in parallel; the hypertonic medium comprises the following components (g/L): (NH) 4 ) 2 SO 4 ，20g/L；Uera，5g/L；KH 2 PO 4 ，1g/L；K 2 HPO 4 ·3H 2 O，1.3g/L；MOPS，42g/L；CaCl 2 ，0.01g/L；FeSO 4 ·7H 2 O，0.01g/L；MnSO 4 ·H 2 O，0.01g/L；ZnSO 4 ·7H 2 O，0.001g/L；CuSO 4 ，0.0002g/L；NiCl·6H 2 O，0.00002g/L；MgSO 4 ·7H 2 O,0.25g/L; protocatechuic acid, 0.03g/L; VB1,0.0001g/L; biotin, 0.0002g/L; yeast powder, 2g/L; glucose, 40g/L; lysine, 175g/L; h 2 SO 4 Adjusting the pH to 7.1-7.2; the non-hypertonic medium is a medium in which lysine is not added.
In the above proteins, the tag refers to a polypeptide or protein which is fused and expressed together with the target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein; the protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
The second surface provides an application of a functional gene of microorganism osmotic pressure tolerance in improving osmotic pressure tolerance and/or amino acid yield of a starting strain, wherein the functional gene of osmotic pressure tolerance is protein shown in any one of the following (A1) to (A4):
(A1) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 2-16;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in any one of SEQ ID NO.2-16 and has the same function as (A1);
(A3) A protein having 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence defined in any one of (A1) or (A2), and having the same function as (A1);
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
Preferably, the microorganism is a microorganism of the genus corynebacterium; further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
In a specific embodiment of the invention, the corynebacterium glutamicum is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any one of the above.
Preferably, the amino acid is lysine or glutamic acid.
In a third aspect, there is provided a recombinant strain having improved osmotic pressure tolerance and/or improved amino acid production, wherein the recombinant strain has an enhanced activity of an osmotic pressure tolerance functional gene relative to a starting strain, and wherein the osmotic pressure tolerance functional gene is a protein as shown in any one of the following (A1) to (A4):
(A1) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 2-16;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in any one of SEQ ID NO.2-16 and has the same function as (A1);
(A3) A protein having 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence defined in any one of (A1) or (A2), and having the same function as (A1);
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
Preferably, the recombinant strain is a microorganism of the genus corynebacterium. Further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
In a specific embodiment of the invention, the corynebacterium glutamicum is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any one of the above.
Preferably, the amino acid is lysine or glutamic acid.
In a fourth aspect, there is provided the use of a recombinant strain according to the third aspect for increasing the osmotic pressure tolerance of a starting strain and/or for the production of amino acids.
Preferably, the amino acid is lysine or glutamic acid.
In a fifth aspect, there is provided a method for constructing a recombinant strain according to the third aspect, comprising overexpressing an osmotically tolerant functional gene in a starting strain, the osmotically tolerant functional gene being a protein as shown in any one of the following (A1) to (A4):
(A1) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 2-16;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in any one of SEQ ID NO.2-16 and has the same function as (A1);
(A3) A protein having 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence defined in any one of (A1) or (A2), and having the same function as (A1);
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
Preferably, the strain is a microorganism of the genus Corynebacterium. Further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
In a specific embodiment of the invention, the corynebacterium glutamicum is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any one of the above.
Preferably, the amino acid is lysine or glutamic acid.
In a sixth aspect, there is provided a method of increasing the tolerance of a microorganism to osmotic pressure and/or amino acid production, the method comprising: enhancing the activity of an osmotic pressure tolerance functional gene in a starting bacterium, wherein the osmotic pressure tolerance functional gene is any one of the following proteins (A1) to (A4):
(A1) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 2-16;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in any one of SEQ ID NO.2-16 and has the same function as (A1);
(A3) A protein having 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence defined in any one of (A1) or (A2), and having the same function as (A1);
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
Preferably, the microorganism is a microorganism of the genus corynebacterium; further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
In a specific embodiment of the invention, the corynebacterium glutamicum is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any one of the above.
Preferably, the amino acid is lysine or glutamic acid.
In a seventh aspect, the present invention provides a method for producing an amino acid, the method comprising producing a target amino acid using the amino acid producing strain of the third aspect, optionally comprising the step of isolating the target amino acid from a fermentation broth.
Preferably, the amino acid is lysine or glutamic acid.
The invention has the beneficial effects that:
the invention provides a method for rapidly and effectively identifying osmotic pressure tolerance functional genes in a large scale, by the method, the genes in a whole genome scale can be rapidly screened and functionally identified, the flux of gene functional identification is greatly increased, and the method has the advantages of simplicity, rapidness, large flux and high hit rate, so that the identification and excavation of the functional genes are more efficient.
Through over-expression of 15 functional genes for improving the osmotic pressure tolerance of microorganisms in corynebacterium glutamicum, the invention discovers that the over-expression can improve the tolerance of strains to high osmotic pressure and promote the growth of original strains, thereby improving the amino acid yield of the strains.
Drawings
FIG. 1.3048 preliminary screening results for genes;
FIG. 2. Re-screening results for lysine high osmotic pressure tolerance gene;
FIG. 3 growth of lysine high osmotic pressure tolerance gene under sodium sulfate hypertonic condition;
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.
Terminology and definition
The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or specification may refer to "one" but may also refer to "one or more", "at least one" and "one or more".
As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps.
Throughout this application, the term "about" means: one value includes the standard deviation of the error of the device or method used to determine the value.
Although the disclosure supports the definition of the term "or" as being inclusive of alternatives and "and/or", the term "or" in the claims means "and/or" unless expressly indicated otherwise as being exclusive of each other, as defined by the alternatives or alternatives.
The "starting strain" and the "wild strain" in the present invention may be microorganisms that are found in nature without artificial modification. In addition, wild-type microorganisms also include modified and engineered microorganisms derived therefrom. For example, in the present invention, the strain may be regarded as a starting strain as long as the Cgl2496 protein is not introduced into the strain or the expression level thereof is enhanced.
The term "recombinant strain" according to the present invention is an engineered strain obtained by recombinant means. Embodiments include, but are not limited to, increasing the copy number of a protein-encoding gene, modifying the sequence of a protein-encoding gene, introducing exogenous recombinant genes, and the like.
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 "recombinant polynucleotide" belongs to one of the "polynucleotides".
The term "coding 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 coding 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 Coding gene contains a CDS Sequence (Coding Sequence) which directs the production of mRNA encoding the protein. Illustratively, the protein-encoding gene of the present invention is a gene encoding an amino acid efflux protein.
The terms "sequence homology" and "percent homology" 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 homology between two or more polynucleotides or polypeptides can 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 origins 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 a polynucleotide or polypeptide. For example, percent homology 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.
In some embodiments, two or more sequences or subsequences have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide "sequence homology" or "percent homology" when compared and aligned for maximum correspondence using a sequence comparison algorithm or as measured by visual inspection. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the compared biopolymers (e.g., polynucleotides).
As used in this disclosure, the term "expression" includes any step involving RNA production and protein production, including, but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
As used in this disclosure, the term "vector" refers to a DNA construct containing 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 disclosure 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. In the present disclosure, "recombinant expression vector" and "recombinant vector" may be used interchangeably.
In some embodiments, "enhancement of protein activity" has a meaning well known to those skilled in the art, including enhancement of expression levels of protein-encoding genes, enhancement of transcription levels of protein-encoding genes, enhancement of protein activity, and the like, including recombinant strains engineered by the following genetic engineering methods: introducing a strong promoter and a strong ribosome binding site into the strain; introducing a recombinant expression vector of the non-integral protein; introducing a recombinant expression vector of the chromosomal integrated protein; altering the promoter, translational regulatory region or coding region codon of the coding gene to enhance transcription or translation; changing the coding gene sequence to enhance mRNA stability or stabilize the structure of the coding protein; or any other means of modifying the coding region of the gene and its adjacent upstream and downstream regions to enhance its activity.
The term "overexpression" as used herein has the meaning conventionally understood by those of skill in the art and may be implemented by methods known in the art, including, but not limited to, such as: inserting a polynucleotide comprising a polynucleotide sequence encoding a protein into a chromosome, and/or cloning the polynucleotide into a vector, and/or directly increasing the copy number of the polynucleotide upstream of the chromosome, and/or engineering a polynucleotide promoter having the encoding of the protein to enhance transcription initiation rate, and/or modifying transcription of the polynucleotide encoding the protein to enhance its activity, and/or modifying the translational regulatory sequences of messenger RNAs carrying the polynucleotide encoding the protein to enhance translational strength, and/or modifying the polynucleotide itself encoding the protein to enhance mRNA stability, protein stability, release of feedback inhibition of the protein, and the like, as well as any known methods that can introduce protein activity.
The term "amino acid" or "L-amino acid" in the present invention refers to the basic building block of a protein in which amino and carboxyl groups are bound to the same carbon atom. Illustratively, 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. In a specific embodiment of the present invention, the amino acid is lysine or glutamic acid.
The "amino acid producing strain" in the present invention means any type of strain that can be used for producing amino acids, and its strain sources include, but are not limited to, escherichia (Erwinia), serratia (Serratia), providia (Providencia), enterobacter (Enterobacter), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium) or Corynebacterium (Corynebacterium). In some embodiments, the amino acid producing strain is derived from corynebacterium glutamicum. In some preferred embodiments, the amino acid producing strain is derived from corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any of the foregoing.
In some embodiments, for lysine producing strains, there may be strains expressing the aspartokinase LysC released from feedback inhibition on the basis of Corynebacterium glutamicum ATCC 13032, which has been reported in the prior art as LysC released from feedback inhibition T311I 、LysC S301F 、LysC L301K 、LysC L301M 、LysC FBR 、LysC I293Y 、LysC I293Q 、LysC D294F 、LysC T307G Etc. The lysine-producing strain may be another strain having lysine-producing ability.
In some embodiments, the lysine producing strain may further include, but is not limited to, one or more genes selected from the group consisting of:
a. an adhE gene encoding alcohol dehydrogenase;
b. an ackA gene encoding acetate kinase;
c. a pta gene encoding phosphoacetyltransferase;
d. the ldhA gene encoding lactate dehydrogenase;
e. the focA gene encoding a formate transport protein;
f. a pflB gene encoding pyruvate formate lyase;
g. a poxB gene encoding pyruvate oxidase;
h. the thrA gene encoding aspartokinase I/homoserine dehydrogenase I bifunctional enzyme;
i. thrB gene encoding homoserine kinase;
j. an ldcC gene encoding lysine decarboxylase; and
h. the cadA gene encoding lysine decarboxylase.
In some embodiments, the lysine-producing strain may further include, but is not limited to, one or more genes selected from the group consisting of:
a. The dapA gene encoding a dihydrodipyridine synthetase that releases feedback inhibition of lysine;
b. the dapB gene encoding a dihydrodipicolinate reductase;
c. a ddh gene encoding diaminopimelate dehydrogenase;
d. dapD encoding a tetrahydropyridine dicarboxylic acid succinylase and dapE encoding a succinyldiaminopimelate deacylase;
e. an asd gene encoding aspartate-semialdehyde dehydrogenase;
f. a ppc gene encoding a phosphoenolpyruvate carboxylase;
g. a pntAB gene encoding a nicotinamide adenine dinucleotide transhydrogenase;
i. the transporter lysE gene encoding lysine.
In some embodiments, for the glutamic acid producing strain, there may be a strain obtained by engineering on the basis of corynebacterium glutamicum ATCC 13032, corynebacterium glutamicum ATCC13869, such engineering including, but not limited to, enhancement or overexpression of one or more genes selected from the group consisting of:
a. yggB gene encoding mechanically sensitive channel protein;
b. the fxpk gene encoding phosphoketolase;
c. the pyc gene encoding pyruvate carboxylase;
d. a gdh gene encoding glutamate dehydrogenase;
e. a gene encoding carbonic anhydrase.
In some embodiments, the glutamic acid-producing strain may further include, but is not limited to, one or more genes selected from the group consisting of:
a. An odhA gene encoding alpha-ketoglutarate dehydrogenase;
b. an amtR gene encoding a transcriptional regulatory gene;
c. acnR gene encoding a transcriptional repressor.
The term "transformation" according to the invention has the meaning generally understood by the person skilled in the art, i.e.the process of introducing exogenous DNA into a strain. The transformation method includes any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl) 2 ) Precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, and lithium acetate-DMSO.
The culture of the strain of the present invention may be carried out according to a conventional method in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, fed-batch culture, etc., and various culture conditions such as temperature, time, pH of the medium, etc., may be appropriately adjusted depending on the actual situation.
The "high osmotic pressure" of the present invention may be a high concentration of Na in the medium 2 SO 4 、K 2 SO 4 Inorganic salt ions such as KCl, or increased concentrations as fermentation time increases, products such as lysine and glutamic acid in the fermentation broth or certain intermediate metabolites accumulate, or increased concentrations due to substrate addition or run-on (e.g., substrates such as ammonium sulfate and glucose), or any other concentrations that may occur in the fermentation broth that may cause osmotic agents. In a specific embodiment, the high osmotic pressure means that the medium is added 175g/L lysine or 85g/L to 156g/L sodium sulfate.
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: construction of a full genome monogenic overexpression plasmid of Glutamicum ATCC13032
The invention firstly constructs a functional gene screening method based on a whole genome single gene overexpression library, which is used for rapidly screening functional genes in high throughput. The specific operation is as follows:
construction of the C.glutamicum ATCC13032 Whole genome monogenic overexpression plasmid was performed on the basis of the derived plasmid pEC-ccdB plasmid of pEC-XK99E plasmid (GenBank: AY 219683.1), P induced by IPTG trc The promoter controls the expression of the gene. The pEC-ccdB plasmid is obtained by transformation on the basis of pEC-XK99E plasmid, the original core skeleton is maintained, firstly, in order to realize efficient cloning, bsaI restriction enzyme cutting sites on the plasmid are removed through point mutation, and then a common efficient cloning screening marker ccdB expression frame is added at the original cloning site; at the same time at P trc The RBS sequence required for gene expression is added behind the promoter, and the plasmid sequence is shown as SEQ ID NO: 1. The plasmid pEC-ccdB was subjected to single cleavage by BsaI endonuclease to obtain a linearized vector. The homologous primers Cglxxxx-F and Cglxxxx-R containing the backbone portion of the plasmid were designed based on the sequence information of plasmid pEC-ccdB. The coding gene fragment was amplified using the ATCC13032 genome as a template according to the reported genomic sequence of Corynebacterium glutamicum ATCC13032 (Genbank ID: NC-003450.3). The linearization vector and the target gene fragment are recovered and then are connected through a one-step recombination kit of Northenzan, then are transformed into E.coli Trans-T1 (Beijing full-type gold biotechnology Co., ltd.), are coated on LB solid medium containing 25 mug/mL kanamycin, are cultured overnight at 37 ℃, PCR verification is carried out on the obtained transformant by utilizing general primers PTRC99C-F and PBV220-R of plasmid pEC-ccdB, and sequencing verification is carried out by a sequencing company, and the correct recombinant plasmid is named pEC-cglxxx. At the same time, a reference substance without cloning any gene sequence in the middle is constructed Pellet pEC-0. The invention relates to a glutamicum ATCC13032 whole genome with more than 3000 genes, which directly relates to a gene cgl0063 amino acid sequence as shown in SEQ ID NO:2 is shown in the figure; cgl0470 amino acid sequence as shown in SEQ ID NO:3 is shown in the figure; the cgl0917 amino acid sequence is shown in SEQ ID NO:4 is shown in the figure; the cgl0923 amino acid sequence is shown in SEQ ID NO:5 is shown in the figure; the cgl1010 amino acid sequence is shown in SEQ ID NO:6 is shown in the figure; the cgl1118 amino acid sequence is shown in SEQ ID NO: shown in figure 7; the cgl1472 amino acid sequence is shown in SEQ ID NO: shown as 8; cgl1994 amino acid sequence as set forth in SEQ ID NO: shown as 9; the cgl2392 amino acid sequence is shown in SEQ ID NO:10 is shown in the figure; the cgl2496 amino acid sequence is shown in SEQ ID NO: 11; the cgl2610 amino acid sequence is shown in SEQ ID NO: shown at 12; the cgl2806 amino acid sequence is shown in SEQ ID NO: 13; the amino acid sequence of cgl2911 is shown in SEQ ID NO: 14; the cgl2998 amino acid sequence is shown in SEQ ID NO: 15; the cgl3003 amino acid sequence is shown in SEQ ID NO: shown at 16. The 15 genes directly related to the present invention, which contain the homologous primers of the plasmid backbone portion are shown in Table 1.
TABLE 1
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EXAMPLE 2.C.glutamicum ATCC13032 construction of Whole genome Single Gene overexpression library
C.glutamicum ATCC13032 competent cells were prepared according to the method reported in the conventional literature, 3000 over-expression plasmids and pEC-0 control plasmids successfully constructed in example 1 were transferred into C.glutamicum ATCC13032 competent cells, respectively, 1mL of TSB medium preheated at 46℃was added, incubated at 46℃for 6min, incubated at 30℃for 2h, and spread on TSB solid medium containing 25. Mu.g/mL kanamycin, and cultured at 30℃for 1 day to obtain transformants. The TSB liquid culture medium comprises the following components (g/L): glucose, 5g/L; yeast powder, 5g/L; soybean peptone, 9g/L; urea, 3g/L; succinic acid, 0.5g/L; k (K) 2 HPO 4 ·3H 2 O，1g/L；MgSO 4 ·7H 2 O,0.1g/L; biotin, 0.01mg/L; vitamin B1,0.1mg/L; MOPS,20g/L. The TSB solid medium was supplemented with 15g/L agar powder. The transformants were PCR verified and sequencing verified using primers PTRC99C-F and PBV220-R, and the correct recombinant strain obtained was designated ATCC13032 (pEC-cglxxx) and control strain ATCC13032 (pEC-0).
EXAMPLE 3 preliminary screening for improved growth Gene by overexpression of lysine under high osmotic pressure conditions
The c.glutamicum ATCC13032 whole genome single gene overexpression library constructed in example 2 was used for a total of 3000 strains, and growth primary screening under lysine high osmotic pressure condition was performed. Both seed and high osmotic pressure cultures were incubated with a MicroScreen fully automated microorganism growth Curve Analyzer (Jieling Co., tianjin) and OD was measured 600 . Seed culture was as follows: the over-expressed strain of the whole genome single gene over-expressed library of C.glutamicum ATCC13032 and the control strain C.glutamicum ATCC13032 (pEC-0) were inoculated into TSB liquid medium containing 25. Mu.g/mL kanamycin, and the culture system was 48-well plates, 400. Mu.L of the stock solution was cultured at 30℃for 8 hours. Transferring 10 μl of the cultured seed solution into lysine hypertonic culture medium respectively, wherein the culture system is 48-well plate, loading 400 μl of the culture solution, culturing at 30deg.C and 800rpm for 96-106 hr with 0.04mM IPTG and 25 μg/mL kana antibiotic, and measuring OD every 1 hr 600 The over-expression gene can be comprehensively screened to improve the growth of the cells under the high osmotic pressure condition. The hypertonic medium comprises the following components (g/L): (NH) 4 ) 2 SO 4 20g/L; urea, 5g/L; KH (KH) 2 PO 4 ，1g/L；K 2 HPO 4 ·3H 2 O，1.3g/L；MOPS，42g/L；CaCl 2 ，0.01g/L；FeSO 4 ·7H 2 O，0.01g/L；MnSO 4 ·H 2 O，0.01g/L；ZnSO 4 ·7H 2 O，0.001g/L；CuSO 4 ，0.0002g/L；NiCl·6H 2 O，0.00002g/L；MgSO 4 ·7H 2 O,0.25g/L; protocatechuic acid, 0.03g/L; VB1,0.0001g/L; biotin, 0.0002g/L; yeast powder, 2g/L; glucose, 40g/L; lysine, 175g/L; h 2 SO 4 The pH is regulated to 7.1-7.2. All over-expressed bacteriaEach time point OD of the strain 600 Calculating the ratio with the control strain, normalizing all batches of data, calculating the average value of 15 continuous maximum values in the growth process of the end point for 30 hours, and judging the genes with 15 values increased or reduced by more than 20% as growth-increasing or reducing genes. As a result, as shown in FIG. 1, all genes showed an average value of 15 consecutive maximum values in the end point growth process for 30 hours, wherein the uppermost dark color point is a gene whose 15 values are each increased by 20% or more, and the lower dark color point represents a gene whose 15 values are each decreased by 20% or more. The total of 29 genes with improved growth includes the following genes cgl0011, cgl0063, cgl0470, cgl0487, cgl0489, cgl0490, cgl0893, cgl0917, cgl0923, cgl1010, cgl1118, cgl1367, cgl1472, cgl1532, cgl1541, cgl1605, cgl1653, cgl1941, cgl1994, cgl2392, cgl2496, cgl2610, cgl2735, cgl2806, cgl2937, cgl2998, cgl3003, cgl3098.
EXAMPLE 4 high osmotic growth of lysine Gene screening
The 29 genes with increased growth obtained by the preliminary screening in example 3 were subjected to a double screening for lysine high osmotic pressure and non-high osmotic pressure. The resulting 29 gene-overexpressed strain and C.glutamicum ATCC13032 (pEC-0) control strain were inoculated into a TSB liquid medium containing 25. Mu.g/mL kanamycin, respectively, and the culture system was a 48-well plate, 400. Mu.L of the liquid was filled, and cultured at 30℃for 8 hours. According to the initial OD 600 Transferring to non-hypertonic and hypertonic culture medium at 30 deg.C and 800rpm for 96-106 hr with culture system of 48-well plate containing 400 μl, adding 0.04mM IPTG and 25 μg/mL kana antibiotic, and detecting OD every 2 hr 600 The experiment was set up in three parallels. Culture and OD determination were still performed using a MicroScreen fully automated microorganism growth curve Analyzer 600 . The hypertonic medium comprises the following components (g/L): (NH) 4 ) 2 SO 4 20g/L; urea, 5g/L; KH (KH) 2 PO 4 ，1g/L；K 2 HPO 4 ·3H 2 O，1.3g/L；MOPS，42g/L；CaCl 2 ，0.01g/L；FeSO 4 ·7H 2 O，0.01g/L；MnSO 4 ·H 2 O，0.01g/L；ZnSO 4 ·7H 2 O，0.001g/L；CuSO 4 ，0.0002g/L；NiCl·6H 2 O，0.00002g/L；MgSO 4 ·7H 2 O,0.25g/L; protocatechuic acid, 0.03g/L; VB1,0.0001g/L; biotin, 0.0002g/L; yeast powder, 2g/L; glucose, 40g/L; lysine, 175g/L; h 2 SO 4 The pH is regulated to 7.1-7.2. The non-hypertonic medium is a medium to which lysine is not added. 3 parallel samples of all strains OD at each time point 600 The mean and standard deviation were calculated separately and the growth curves for each strain were plotted for non-hypertonic and hypertonic conditions. Sample overexpressing strain and control strain OD at each time point of the 30h end point growth process were analyzed again 600 Significance of difference (T-test analysis), P if hypertonic conditions are satisfied for 15 consecutive time points and above<0.05, and the overexpression strain OD of these spots 600 The average value is larger than that of the control strain, so that the over-expression of the gene can improve the strain growth under the condition of lysine hyperpermeability. The 29 genes obtained were initially screened, and only 15 genes after rescreening met the above requirements, and the growth curves for non-hypertonic and hypertonic conditions are shown in FIG. 2, and it can be seen that cgl0063, cgl0470, cgl0917, cgl0923, cgl1010, cgl1118, cgl1472, cgl1994, cgl2392, cgl2496, cgl2610, cgl2806, cgl2911, cgl2998 and cgl3003 still improved strain growth under hypertonic conditions; meanwhile, the over-expression of the cgl3003 can not only improve the growth of the strain under the hypertonic condition, but also improve the growth of the strain under the non-hypertonic condition; other gene overexpression only enhances strain growth under hypertonic conditions. Under the condition of lysine hypertonicity, the maximum enhancement amplitude of the gene meeting the above criteria at the time point of the growth enhancement phase is as follows: the maximum improvement of cgl0063 is 16% at 68h, cgl0470 is 24% at 72h, cgl0917 is 18% at 66h, cgl0923 is 14% at 72h, cgl1010 is 36% at 72h, cgl1118 is 52% at 42h, cgl1472 is 33% at 72h, cgl1994 is 16% at 96h, cgl2392 is 25% at 72h, cgl2496 is 43% at 90h, cgl2610 is 51% at 90h, cgl2806 is 53% at 90h, cgl2911 is 28% at 103h, cgl2998 is 36% at 104h, cgl3003 was increased by a maximum of 23% at 60 h.
EXAMPLE 5.15 verification of the tolerance of the hypertonic-resistant genes to sodium sulfate osmotic pressure
The 15 gene-overexpressing strains obtained in example 4 and ATCC13032 (pEC-0) control strains were inoculated into TSB liquid medium containing 25. Mu.g/mL kanamycin, respectively, and cultured at 30℃for 8 hours, according to the initial OD 600 Transferring into sodium sulfate hypertonic culture medium at 0.1, culturing in 48-well plate with 400 μl of IPTG and 25 μg/mL kana antibiotic at 0.04mM per well, culturing at 30deg.C and 800rpm for 96-120 hr with MicroScreen full-automatic microorganism growth curve analyzer, and detecting OD every 2 hr 600 Three experiments are performed in parallel, and the functions of 15 genes in the high osmotic pressure response and tolerance of corynebacterium glutamicum are comprehensively characterized. The hypertonic medium was 156g/L sodium sulfate added to the non-hypertonic medium of example 3. As shown in FIG. 3, the data analysis was performed using the same criteria as in example 4, and it was found that 15 genes also improved growth under sodium sulfate hypertonic conditions. The above results show that the 15 gene overexpression obtained by rescreening has a beneficial effect on improving the high osmotic pressure tolerance of corynebacterium glutamicum. Under sodium sulfate hypertonic conditions, in the growth enhancement phase, cgl0063 was maximally 25% enhancement at 64h, cgl0470 was maximally 40% enhancement at 118h, cgl0917 was maximally 37% enhancement at 50h, cgl0923 was maximally 44% enhancement at 26h, cgl1010 was maximally 46% enhancement at 26h, cgl1118 was maximally 11% enhancement at 60h, cgl1472 was maximally 36% enhancement at 26h, cgl1994 was maximally 20% enhancement at 118h, cgl2392 was maximally 63% enhancement at 26h, cgl2496 was maximally 60% enhancement at h, cgl2610 was maximally 52% enhancement at 26h, cgl2806 was maximally 28% enhancement at 26h, cgl2911 was maximally 87% enhancement at 26h, cgl2998 was maximally 83% enhancement at 26h, and cgl3003 was maximally 92% enhancement at 26 h.
EXAMPLE 6.15 validation of lysine production by the high-penetration resistant Gene
pEC-cgl0063, pEC-cgl0470, pEC-cgl0917, pEC-cgl0923, pEC-cgl1010, pEC-cgl1118, pEC-cgl1472, pEC-cgl1994, pEC-cgl2392, pEC-cgl2496, pEC-cgl2610, pEC-cgl2806, pEC-cgl2911, pEC-cgl2998, pEC-cgl3003 and control pEC-0 plasmids were introduced into lysine-producing strain AHP-3 (see CN 113249347A) by electrotransformation, respectively, to obtain AHP-3 (pEC-cgl 0063), AHP-3 (pEC-cgl 0470), AHP-3 (pEC-cgl 0917), AHP-3 (pEC-cgl 0923), AHP-3 (pEC-cgl 1010), AHP-3 (pEC-cgl 1118), AHP-3 (pEC-cgl 1472), AHP-3 (pEC-cgl 1994), AHP-3 (pEC-cgl 2392), AHP-3 (pEC-cgl 2496), AHP-3 (pEC-cgl 2610), AHP-3 (pEC-cgl 2806), AHP-3 (pEC-cgl 292911), AHP-3 (pEC-cgl 98), AHP-3 (pEC-cgl 3) and pEC-300 strain (pEC-300). The above overexpression strain and the control strain were inoculated into TSB liquid medium containing 25. Mu.g/mL kanamycin, respectively, for seed culture, the culture system was 800. Mu.L of 24-well plate loading liquid, and the culture was carried out at 30℃and 800rpm using a high throughput shaker (Chu instruments Co., shanghai) for about 8 hours. The seed solution is subjected to initial OD 600 To 0.1, the individual strains were transferred to a fermentation medium supplemented with 0.04mM IPTG and 25. Mu.g/mL kana antibiotics, the culture system was 800. Mu.L per well of a 24-well plate, and each strain was grown in triplicate using a high throughput shaker at 30 ℃. The fermentation medium comprises the following components (g/L): glucose, 60g/L; yeast powder, 1g/L; soybean peptone, 1g/L; naCl,1g/L; ammonium sulfate, 1g/L; urea, 8g/L; k (K) 2 HPO 4 ·3H 2 O，1g/L；MgSO 4 ·7H 2 O，0.45g/L；FeSO 4 ·7H 2 O,0.05g/L; biotin, 0.4mg/L; VB1,0.1mg/L; MOPS,40g/L; na (Na) 2 SO 4 85g/L; the initial pH7.2 was adjusted with ammonia water, and the L-lysine production was measured at the time of fermentation for 66 hours. As shown in Table 2, the OD of growth was in addition to AHP-3 (pEC-cgl 1118) 600 The values were not significantly different from the control AHP-3 (pEC-0), with about 10% improvement in AHP-3 (pEC-cgl 0063) and AHP-3 (pEC-cgl 0470) growth, about 20% improvement in AHP-3 (pEC-cgl 0917), AHP-3 (pEC-cgl 0923), AHP-3 (pEC-cgl 1010), AHP-3 (pEC-cgl 1472), AHP-3 (pEC-cgl 1994), AHP-3 (pEC-cgl 2392), AHP-3 (pEC-cgl 2496), AHP-3 (pEC-cgl 2610), AHP-3 (pEC-cgl 2806), AHP-3 (pEC-cgl 2911), AHP-3 (pEC-cgl 2998) and AHP-3 (pEC-cgl 3003) growth, wherein the highest of AHP-3 (pEC-96) is reached at least 48%. In terms of lysine production, the level of AHP-3 (pEC-cgl 0063) was 42% higher than that of control AHP-3 (pEC-0), AHP-3 (pEC-cgl 0470), AHP-3 (pEC-cgl 0917), AHP-3 (pEC-cgl 0923),AHP-3 (pEC-cgl 1010), AHP-3 (pEC-cgl 1118), AHP-3 (pEC-cgl 1472), AHP-3 (pEC-cgl 1994), AHP-3 (pEC-cgl 2392), AHP-3 (pEC-cgl 2496), AHP-3 (pEC-cgl 2610), AHP-3 (pEC-cgl 2806), AHP-3 (pEC-cgl 2911), AHP-3 (pEC-cgl 2998) and AHP-3 (pEC-cgl 3003) may be increased by more than 50%, wherein AHP-3 (pEC-cgl 1472), AHP-3 (pEC-cgl 1994), AHP-3 (pEC-cgl 2610) and AHP-3 (pEC-cgl 2806) may be increased by more than 100%.
TABLE 2 Effect of osmotic tolerance Gene overexpression on lysine production
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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 high-throughput identification method of a functional gene of osmotic pressure tolerance of a microorganism, which is characterized by comprising the following steps:
1) Constructing a single gene overexpression library based on a microbial whole genome, and constructing a control plasmid strain without cloning any gene sequence in the middle;
2) Primary screening of over-expression growth improvement genes under high osmotic pressure conditions;
3) Re-screening of the over-expressed growth-promoting genes under high osmotic pressure and non-high osmotic pressure conditions;
preferably, the step 2) is specifically to transfer the microorganism whole genome monogenic overexpression strain constructed in the step 1) and seed solution containing the control plasmid strain into lysine hypertonic culture medium respectively, and culture and OD determination are carried out by adopting a MicroScreen full-automatic microorganism growth curve analyzer 600 OD at each time point of all over-expressed strains 600 Calculating ratio with control strain, and collectingCarrying out unification processing on all batch data, calculating an average value of 15 continuous maximum values in the final growth process for 30 hours, and judging genes with 15 values improved by more than 20% as growth improvement genes;
further preferably, the step 3) specifically comprises the steps of carrying out rescreening of lysine high osmotic pressure and non-high osmotic pressure on the strain with the over-expressed growth enhancing gene obtained by the preliminary screening in the step 2) and the strain containing the control plasmid; cultivation and determination of OD Using MicroScreen fully automatic microorganism growth Curve Analyzer 600 3 parallel samples of all strains OD at each time point 600 Calculating average value and standard deviation respectively, and drawing a growth curve of each strain under non-hypertonic and hypertonic conditions; the strain overexpressed by the sample at each time point during the 30h end point growth process was analyzed again with the strain OD containing the control plasmid 600 Significance of difference (T-test analysis), P if hypertonic conditions are satisfied for 15 consecutive time points and above<0.05, and the overexpression strain OD of these spots 600 The average value is larger than that of the control strain, so that the over-expression of the gene can improve the strain growth under the condition of lysine hyperpermeability;
more preferably, the culture system of the step 2) is a 48-well plate, 400 mu L of loading liquid is filled, the culture medium contains 0.04mM IPTG and 25 mu g/mL kana antibiotics, the culture is carried out at 30 ℃ and 800rpm for 96-106 hours, and the components of the hypertonic culture medium are (g/L): (NH) 4 ) 2 SO 4 ，20g/L；Uera，5g/L；KH 2 PO 4 ，1g/L；K 2 HPO 4 ·3H 2 O，1.3g/L；MOPS，42g/L；CaCl 2 ，0.01g/L；FeSO 4 ·7H 2 O，0.01g/L；MnSO 4 ·H 2 O，0.01g/L；ZnSO 4 ·7H 2 O，0.001g/L；CuSO 4 ，0.0002g/L；NiCl·6H 2 O，0.00002g/L；MgSO 4 ·7H 2 O,0.25g/L; protocatechuic acid, 0.03g/L; VB1,0.0001g/L; biotin, 0.0002g/L; yeast powder, 2g/L; glucose, 40g/L; lysine, 175g/L; h 2 SO 4 Adjusting the pH to 7.1-7.2;
step 3) the culture system is that 400 mu L of 48-well plate filling liquid is added with 0.04mM IPTG and 25 mu g/mL kana antibiotics, the temperature is 30 ℃, the culture is carried out at 800rpm for 96-106h,OD was detected every 2h 600 Three experiments were set up in parallel; the hypertonic medium comprises the following components (g/L): (NH) 4 ) 2 SO 4 ，20g/L；Uera，5g/L；KH 2 PO 4 ，1g/L；K 2 HPO 4 ·3H 2 O，1.3g/L；MOPS，42g/L；CaCl 2 ，0.01g/L；FeSO 4 ·7H 2 O，0.01g/L；MnSO 4 ·H 2 O，0.01g/L；ZnSO 4 ·7H 2 O，0.001g/L；CuSO 4 ，0.0002g/L；NiCl·6H 2 O，0.00002g/L；MgSO 4 ·7H 2 O,0.25g/L; protocatechuic acid, 0.03g/L; VB1,0.0001g/L; biotin, 0.0002g/L; yeast powder, 2g/L; glucose, 40g/L; lysine, 175g/L; h 2 SO 4 Adjusting the pH to 7.1-7.2; the non-hypertonic medium is a medium in which lysine is not added.
2. Use of an osmotically tolerant functional gene obtainable by the method according to claim 1 for increasing osmotically tolerant and/or amino acid production in a starting strain, characterized in that the osmotically tolerant functional gene is a protein as shown in any one of the following:
(A1) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 2-16;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in any one of SEQ ID NO.2-16 and has the same function as (A1);
(A3) A protein having 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence defined in any one of (A1) or (A2), and having the same function as (A1);
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
3. The use according to claim 2, wherein the microorganism is a microorganism of the genus corynebacterium; further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum; more preferably, the corynebacterium glutamicum is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any one of the above;
preferably, the amino acid is lysine or glutamic acid.
4. A recombinant strain with increased osmotic pressure tolerance and/or increased amino acid production, characterized in that the activity of an osmotic pressure tolerance functional gene in the recombinant strain is increased relative to the starting strain, the osmotic pressure tolerance functional gene being a protein as shown in any one of the following:
(A1) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 2-16;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in any one of SEQ ID NO.2-16 and has the same function as (A1);
(A3) A protein having 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence defined in any one of (A1) or (A2), and having the same function as (A1);
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
5. The recombinant strain according to claim 4, wherein the recombinant strain is a microorganism of the genus corynebacterium; further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum;
more preferably, the corynebacterium glutamicum is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any one of the above;
preferably, the amino acid is lysine or glutamic acid.
6. The method for constructing a recombinant strain according to claim 4 or 5, wherein the method comprises overexpressing an osmotically tolerant functional gene in a starting strain, the osmotically tolerant functional gene being any one of the following proteins:
(A1) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 2-16;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in any one of SEQ ID NO.2-16 and has the same function as (A1);
(A3) A protein having 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence defined in any one of (A1) or (A2), and having the same function as (A1);
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
7. The method according to claim 6, wherein the starting strain is a microorganism belonging to the genus Corynebacterium; further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum;
more preferably, the corynebacterium glutamicum is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any one of the above;
preferably, the amino acid is lysine or glutamic acid.
8. A method of increasing the tolerance of a microorganism to osmotic pressure and/or increased amino acid production, the method comprising: enhancing the activity of an osmotic pressure tolerance functional gene in a starting bacterium, wherein the osmotic pressure tolerance functional gene is any one of the following proteins:
(A1) The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 2-16;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in any one of SEQ ID NO.2-16 and has the same function as (A1);
(A3) A protein having 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence defined in any one of (A1) or (A2), and having the same function as (A1);
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
9. The method according to claim 8, wherein the microorganism is a microorganism of the genus corynebacterium; further preferably, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum;
more preferably, the corynebacterium glutamicum is corynebacterium glutamicum ATCC13032, corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 14067, or a derivative strain of any one of the above;
preferably, the amino acid is lysine or glutamic acid.
10. A method for producing an amino acid, characterized in that the method comprises producing a target amino acid using the recombinant strain according to claim 4 or 5, optionally comprising the step of isolating the target amino acid from a fermentation broth; preferably, the amino acid is lysine or glutamic acid.
CN202310256561.7A 2022-04-01 2023-03-16 Osmotic pressure tolerance functional gene identification method and application of functional gene Pending CN116891868A (en)

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