L-amino acid producing microorganism with enhanced cytochrome C activity and L-amino acid production method using same
technical field
[1]
Cross-Citation with Related Applications
[2]
This application claims the benefit of priority based on Korean Patent Application No. 10-2019-0173087 dated December 23, 2019 and Korean Patent Application No. 10-2019-0173088 dated December 23, 2019, and the document of the Korean patent application All content disclosed in is incorporated as a part of this specification.
[3]
Provided are L-amino acid-producing microorganisms with enhanced cytochrome C activity, and methods for producing L-amino acids using the microorganisms.
[4]
background
[5]
The microorganism of the genus Corynebacterium is a gram-positive microorganism that is widely used for the production of L-amino acids. L-amino acids, particularly L-lysine, are used in animal feed, human pharmaceuticals and cosmetics industries, and are mainly produced by fermentation using a Corynebacterium strain.
[6]
Many attempts have been made to improve the method for producing L-amino acids using Corynebacterium sp. strains. Among them, there is a study to improve Corynebacterium strains that produce L-amino acids by destroying or attenuating expression of specific genes using recombinant DNA technology. In addition, by amplifying the genes involved in the biosynthesis of each L-amino acid to study the effect on the production of L- amino acid, research has been conducted to improve the L- amino acid producing Corynebacterium strain.
[7]
On the other hand, in the lysine production industry through fermentation, the production of high concentrations of lysine and increase in the lysine production capacity of microorganisms are important factors. Therefore, efforts have been made to improve the lysine production capacity of microorganisms and to continuously maintain them during fermentation and culture. However, due to various internal or external factors that inhibit the activity of microorganisms, there is a problem in that it is difficult to maintain the lysine production ability until the latter half of culture.
[8]
Therefore, L- amino acids, for example, the production capacity of L- lysine is improved and the development of a continuously sustainable strain is required.
[9]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[10]
One example provides an L-amino acid-producing microorganism with enhanced cytochrome C (Cytochrome C) activity. For example, the enhancement of cytochrome C activity may be due to introduction of a gene encoding cytochrome C. For example, the gene encoding the cytochrome C may be a foreign gene.
[11]
Another example provides a method for producing L-amino acids, comprising the step of culturing the L- amino acid producing microorganism.
[12]
Another example provides a composition for enhancing L-amino acid production or L-amino acid production in a microorganism, including a cytochrome C coding gene, a recombinant vector including the gene, or both.
[13]
means of solving the problem
[14]
In one embodiment provided herein, the effect on lysine production by amplifying a gene for the purpose of increasing the lysine production capacity of the microorganisms of the genus Corynebacterium is confirmed, and based on this, a strain improvement technology for amino acid production is proposed. In general, as a method for increasing the production capacity of amino acids such as lysine, there may be mentioned methods of improving the yield of amino acids such as lysine possessed by the strain, or increasing the production (productivity) of amino acids such as lysine per hour. In particular, the productivity of amino acids such as lysine may be affected by various factors such as components of the fermentation medium, osmotic pressure of the fermentation medium, temperature, agitation rate, and oxygen supply rate. For example, due to problems such as stress by various substances and metabolites present in the fermentation broth, oxygen supply decrease due to increase in microbial cells, and physical conditions such as temperature and agitation speed, the lysine production ability of microorganisms and cellular activity of microorganisms continuously decreases. In an example of the present specification, there is provided a strain improvement technique that overcomes the stress caused by such various factors and maintains the target product production activity of the microorganism until the latter half of fermentation.
[15]
Hereinafter, it will be described in more detail.
[16]
One example provides an L-amino acid-producing microorganism with enhanced cytochrome C (Cytochrome C) activity.
[17]
As used herein, the term "L-amino acid-producing microorganism" refers to a case in which a microorganism having L-amino acid-producing ability has increased L-amino acid-producing ability by enhancing cytochrome C activity and/or does not have L-amino acid-producing ability. It can be used to mean a case in which microorganisms have L-amino acid production ability by enhancing cytochrome C activity. As used herein, “microorganism” encompasses single-celled bacteria, and may be used interchangeably with “cell”.
[18]
The L-amino acid may be L-lysine.
[19]
In the present specification, in order to distinguish the microorganism before the cytochrome C activity is enhanced from the "L-amino acid producing microorganism" to which the cytochrome C activity is enhanced and L-amino acid production capacity is increased or L-amino acid production ability is conferred, the cytochrome C activity A microorganism before the chromium C activity is enhanced can be expressed as a host microorganism.
[20]
In one example, the host microorganism may be selected from all microorganisms having the ability to produce L-amino acids (eg, L-lysine). In one example, the host microorganism may be a microorganism having L-lysine-producing ability naturally or a microorganism having L-lysine-producing ability by introducing a mutation into a parent strain having no or significantly less L-lysine-producing ability.
[21]
In one example, the host microorganism is any gram-positive bacteria having L-lysine-producing ability by introducing a mutation into a microorganism having naturally L-lysine-producing ability or a parent strain having no or significantly less L-lysine-producing ability, such as Corynebacter The genus　 Corynebacterium may be at least one selected from the group consisting of microorganisms and the genus Escherichia microorganisms. The Corynebacterium genus microorganism is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Brevibacterium lactofermentum ( Brevibacterium lactofermentum ), Brevibacterium flabum ( Brevibacterium flavum ), Corynebacterium thermoaminogenes ( Corynebacterium thermoaminogenes ), Corynebacterium efficiens ( Corynebacterium efficiens )) and the like, but is not necessarily limited thereto. For example, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum .
[22]
As used herein, the term "cytochrome C" is derived from bacteria and has an average molecular weight of 15 kDa or less, such as about 8 kDa to about 15 kDa and/or 90 to 150, 100 to 150, 120 to 150, 90 to 125, It is 100 to 125, or 120 to 125 amino acids in length, and refers to cytochrome C of a monomer having membrane-bound properties. In one embodiment, the cytochrome C may be derived from a microorganism of the genus Bacillus, and the wavelength range in which the absorption band (absorbance) of the lowest energy level in the reduced state appears is 550 to 555 nm or 550 to 551 nm, selected from cytochrome C There may be more than one type. In one example, the cytochrome C is at least one selected from the group consisting of cytochrome c-551 (absorbance about 551 nm), cytochrome c-550 (absorbance about 550 nm), etc., derived from microorganisms of the genus Bacillus, such as one, It may be 2 types, or 3 types (the numerical value listed after the above cytochrome c means absorbance). The microorganism of the genus Bacillus may be at least one selected from the group consisting of Bacillus pseudofirmus , Bacillus subtilis , and the like.
[23]
In one example, the cytochrome C, for example, one or more selected from c-551 and cytochrome c-550 may each independently include an amino acid sequence encoded by cccA or cccB. In one embodiment, the cytochrome C is one selected from the group consisting of Bacillus pseudofirmus (eg, Bacillus pseudofirmus OF4, etc.) and/or cytochrome c-551 derived from Bacillus subtilis , cytochrome c-550 derived from Bacillus subtilis, etc. may be more than
[24]
More specifically, the cytochrome C (eg, cytochrome c-551 from Bacillus subtilis ) is a poly containing an amino acid sequence (eg, SEQ ID NO: 16) encoded by cccA (eg, BpOF4_13740 from Bacillus pseudofirmus OF4) It may include a peptide, a polypeptide including an amino acid sequence (eg, SEQ ID NO: 27) encoded by cccB (eg, BpOF4_05495​​derived from Bacillus pseudofirmus OF4), or both.
[25]
In addition, unless otherwise defined, cytochrome C described herein is 20% or more, 30% or more, 40% or more, 50% or more, 55% or more of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 27 described as an example, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater (eg, 60% to 99.5%, 70% to 99.5%, 80% to 99.5%, 85% to 99.5%, 90 % to 99.5%, 91% to 99.5%, 92% to 99.5%, 93% to 99.5%, 94% to 99.5%, 95% to 99.5%, 96% to 99.5%, 97% to 99.5%, 98% to 99.5%, or 99% to 99.5%) can be interpreted as meaning including a protein having sequence homology. As such, the protein having the sequence homology that can be included in the cytochrome C category described herein is (1) the above-described cytochrome C characteristics, such as (a) bacterial origin, (b) an average molecular weight of 15 kDa or less, such as, about 8 kDa to about 15 kDa, and/or 90 to 150, 100 to 150, 120 to 150, 90 to 125, 100 to 125, or 120 to 125 amino acids in length, (c) membrane binding properties, and (d) monomeric properties. have one or more of, and/or (2) an effect by enhancing activity in the microorganism, such as compared to an unmodified microorganism, (e) an L-amino acid (eg,
[26]
In one example, the L-amino acid-producing microorganism with enhanced cytochrome C activity may have an increased ability to produce L-amino acids as compared to the same unmodified microorganism without enhanced cytochrome C activity.
[27]
As used herein, the term “enhancement of cytochrome C activity” refers to any manipulation in which cytochrome C activity is introduced into a microorganism or the cytochrome C activity can be improved compared to the intrinsic activity or activity before modification possessed by the microorganism. there is. The "introduction" means that the activity of cytochrome C, which the microorganism did not originally have, appears naturally or artificially. The "unmodified microorganism" may refer to a host microorganism in which cytochrome C has not been enriched or has not been enriched. The "intrinsic activity" may refer to the activity of cytochrome C originally possessed by the host microorganism before or without cytochrome C enrichment. In the present specification, "unmodified" may be used interchangeably with a form having intrinsic activity in which genetic mutation does not occur.
[28]
For example, the enhancement of cytochrome C activity may include both introducing and enhancing exogenous cytochrome C or enhancing endogenous cytochrome C activity. In one example, the enhancement of cytochrome C activity may be enhanced by introducing exogenous cytochrome C.
[29]
In one example, it can be confirmed that the enhancement of cytochrome C activity in the microorganism is improved compared to the unmodified microorganism in which the sugar consumption rate of the microorganism is not enhanced. In particular, the cytochrome C activity-enhanced microorganism exemplified in one embodiment is similar to the unmodified microorganism and the strain growth (OD value) and / or L-amino acid, for example, L-lysine production yield within a specific section, and the sugar consumption rate may be improved, which suggests that the cytochrome C activity-enhanced microorganism produces more L-amino acids in a shorter time compared to the unmodified microorganisms, thereby improving L-amino acid productivity.
[30]
In one example, the enhancement of cytochrome C activity may be due to increased expression of cytochrome C at the gene (mRNA) level and/or protein level and/or increased activity as cytochrome C, but is limited thereto not.
[31]
In one example, the enhancement of the cytochrome C activity may be by introduction of a gene encoding cytochrome C. By introducing such a gene encoding cytochrome C, the L-amino acid producing ability of the microorganism having the L-amino acid producing ability is increased, or the L-amino acid producing ability can be conferred to the microorganism having the L-amino acid producing ability.
[32]
In one example, the cytochrome C or a gene encoding the same may be derived from (intrinsic) a host microorganism or derived from another microorganism (exogenous). In one embodiment, the enhancement of cytochrome C activity may be performed by introducing a polynucleotide encoding exogenous cytochrome C into a host microorganism. The cytochrome C is the same as described above, and for example, may be cytochrome C (cytochrome c-551) derived from Bacillus pseudofirmus OF4, for example, SEQ ID NO: 16 (eg, encoded by cccA (BpOF4_13740)) Or it may be one represented by the amino acid sequence of SEQ ID NO: 27 (eg, encoded by cccB (BpOF4_05495)).
[33]
In one example, the gene encoding the cytochrome C or the L-amino acid producing microorganism into which the gene is introduced may include a polynucleotide encoding SEQ ID NO: 16, a polynucleotide encoding SEQ ID NO: 27, or both. there is. In one embodiment, the L-amino acid producing microorganism is a microorganism of the genus Corynebacterium comprising a polynucleotide encoding SEQ ID NO: 16, a polynucleotide encoding SEQ ID NO: 27, or both, such as Corynebacterium glue Tamicum ( Corynebacterium glutamicum ) may be. For example, the L-amino acid producing microorganism may be one with accession number KCCM12640P.
[34]
As used herein, a polynucleotide (which may be used interchangeably with "gene") or a polypeptide (which may be used interchangeably with "protein") is "comprising a specific nucleic acid sequence or amino acid sequence, consisting of a specific nucleic acid sequence or amino acid sequence, Or, it is expressed as a specific nucleic acid sequence or amino acid sequence" is an expression that is interchangeable with an equivalent meaning, and may mean that the polynucleotide or polypeptide necessarily includes the specific nucleic acid sequence or amino acid sequence, and the poly Includes "substantially equivalent sequences" in which mutations (deletions, substitutions, modifications, and/or additions) are made to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and/or desired function of the nucleotide or polypeptide is maintained. It can be interpreted as doing (or not excluding that the mutation is introduced).
[35]
In one example, the nucleic acid sequences or amino acid sequences provided herein can be prepared by conventional mutagenesis methods such as direct evolution and/or site-specification to the extent that they retain their original or desired functions. It may include those modified by site-directed mutagenesis or the like. In one embodiment, reference to a polynucleotide or polypeptide “comprising a specific nucleic acid sequence or amino acid sequence” means that the polynucleotide or polypeptide (i) consists of or consists essentially of the specific nucleic acid sequence or amino acid sequence; or (ii) at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95% of the specific nucleic acid sequence or amino acid sequence. or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more (eg, 60% to 99.5%, 70% to 99.5%, 80% to 99.5%, 85% to 99.5%, 90% to 99.5%, 91% to 99.5%, 92% to 99.5%, 93% to 99.5%, 94% to 99.5%, 95% to 99.5%, 96% to 99.5%, 97% to 99.5% , 98% to 99.5%, or 99% to 99.5%) of an amino acid sequence having homology of or essentially including the same, and may mean maintaining the original function and/or the desired function. In the present specification, the original function is a cytochrome C function (in the case of an amino acid sequence),
[36]
The nucleic acid sequence described herein is an amino acid sequence and/or function of a protein expressed from a coding region in consideration of a codon preferred in a microorganism to express the protein (cytochrome C) due to codon degeneracy. Various modifications may be made to the coding region within a range that does not change .
[37]
As used herein, the term "homology" refers to the degree of correspondence with a given nucleic acid sequence or amino acid sequence, and may be expressed as a percentage (%). For homology to nucleic acid sequences, for example, the algorithm BLAST according to the literature (Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (see Methods Enzymol) ., 183, 63, 1990) can be used. Based on such an algorithm BLAST, a program called BLASTN or BLASTX has been developed (refer to http://www.ncbi.nlm.nih.gov).
[38]
In one embodiment, a polynucleotide comprising a specific nucleic acid sequence provided herein comprises a polynucleotide fragment comprising a nucleic acid sequence complementary to the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, as well as the specific nucleic acid sequence. can be interpreted as Specifically, the polynucleotide having the complementarity can be hybridized at a Tm value that can be appropriately adjusted by those skilled in the art depending on the purpose, for example, a Tm value of 55°C, 60°C, 63°C or 65°C, and analyzed under the conditions described below. : These conditions are specifically described in known literature. For example, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more. Genes with high complementarity of at least 98%, 98% or more, 99.5% or more, or 99.9% or more hybridize with each other, and genes with lower complementarity do not hybridize, or general Southern hybridization washing conditions 60 ℃, 1x SSC (saline-sodium citrate buffer), and 0.1% (w / v) SDS (Sodium Dodecyl Sulfate); 60°C, 0.1x SSC, and 0.1% SDS; or washing conditions once, specifically 2 to 3 times, at 68° C., at a salt concentration and temperature equivalent to 0.1x SSC, and 0.1% SDS, and the like. Hybridization requires that two nucleotides have complementary sequences, or mismatches between bases may be allowed depending on the stringency of hybridization. the term "
[39]
If the method for the enhancement at the gene (mRNA) level of cytochrome C during the enhancement of the cytochrome C activity will be described in more detail,
[40]
1) an increase in the number of copies of the polynucleotide encoding the cytochrome C;
[41]
2) modification of the expression control sequence to increase the expression of the polynucleotide, or
[42]
3) A method of deforming to be strengthened by a combination of 1) and 2) above
[43]
and the like may be exemplified, but the present invention is not limited thereto.
[44]
1) The increase in the copy number of the polynucleotide is not particularly limited thereto, but the polynucleotide may be introduced into a host microorganism through a vector or may be performed by being inserted into the chromosome of the host microorganism. For example, the copy number increase may be performed by introducing a polynucleotide encoding an exogenous cytochrome C or a codon-optimized mutant polynucleotide of the polynucleotide into a host microorganism. The exogenous polynucleotide may be used without limitation in its origin or sequence as long as it exhibits the same/similar activity as that of cytochrome C it encodes. The introduction can be carried out by appropriately selecting and/or modifying known transformation methods by those skilled in the art. By the introduction, the introduced polynucleotide is expressed in the host microorganism, thereby making it possible to generate exogenous cytochrome C.
[45]
2) Modification of the expression control sequence so as to increase the expression of the polynucleotide, is applicable to both foreign polynucleotides and endogenous polynucleotides, and is not particularly limited thereto. Deletion, insertion, non-conservative or conservative substitution, mutagenesis of a combination thereof, or replacement with an expression control sequence having stronger activity. The expression control sequence is not particularly limited thereto, but may be one or more selected from the group consisting of a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating the termination of transcription and/or translation. In one embodiment, for increased expression, a strong heterologous promoter can be operably linked upstream of the polynucleotide expression unit instead of the native promoter. Examples of the strong promoter include CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter. More specifically, the strong promoter may be the lysCP1 promoter (WO2009/096689) or the CJ7 promoter (WO2006/065095), which are promoters derived from the genus Corynebacterium, but is not limited thereto.
[46]
Hereinafter, a case in which the above-described cytochrome C-encoding gene is introduced into a host microorganism through a vector or inserted into a chromosome of the host microorganism will be described in more detail. The gene introduction described herein is performed by introducing a foreign gene (derived from a heterologous to and/or from another individual homologous to the host microorganism) into a host cell i) in a form operably linked to a recombinant vector, or ii) the host It can be performed by insertion (eg, random insertion) into the chromosome (genome) of the cell. ii) when inserted into the chromosome (genome) of the host cell, the insertion site is a location that does not affect the growth of the host cell (eg, non-transcriptional spacer (NTS), etc.) and / or random insertion efficiency It may be a position that can be raised (eg, retrotransposon, etc.), but is not limited thereto.
[47]
It may mean that the expression control element (eg, promoter) and the polynucleotide are functionally linked to perform transcription initiation). Operable ligation may be performed using genetic recombination techniques known in the art, for example, but may be performed by conventional site-specific DNA cleavage and ligation, but is not limited thereto.
[48]
The method of transforming the polynucleotide into a host microorganism can be performed by any method of introducing a nucleic acid into a cell (microorganism), and can be performed by appropriately selecting a transformation technique known in the art according to the host microorganism. The known transformation methods include electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol-mediated uptake. ), DEAE-dextran method, cationic liposome method, lipofection, lithium acetate-DMSO method and the like may be exemplified, but is not limited thereto.
[49]
Insertion of the gene into the host cell genome (chromosome) can be performed by appropriately selecting a known method by those skilled in the art, for example, an RNA-guided endonuclease system (RNA-guided endonuclease system; for example, (a) RNA- A guide endonuclease (eg, Cas9 protein, etc.), its coding gene, or a vector comprising the gene; and (b) a guide RNA (eg, single guide RNA (sgRNA), etc.), its coding DNA, or the DNA A mixture (eg, a mixture of RNA-guided endonuclease protein and guide RNA, etc.), a complex (eg, a ribonucleic acid fusion protein (RNP), a recombinant vector (eg, RNA-guided endonuclea) comprising a vector comprising It may be carried out using one or more selected from the group consisting of a vector including the first encoding gene and the guide RNA encoding DNA, etc.), but is not limited thereto.
[50]
In another example, a cytochrome C encoding gene, a recombinant vector comprising the gene, and/or an increase in L-amino acid production capacity and/or L-amino acid production ability in a microorganism of a cell comprising the gene or the recombinant vector The use for is provided.
[51]
One example provides a composition for producing L-amino acids comprising a gene encoding cytochrome C, a recombinant vector including the gene, or a cell including the gene or the recombinant vector.
[52]
Another example may be a cytochrome C-encoding gene, a recombinant vector including the gene, or a composition for L-amino acid production including all of them. The composition for producing L-amino acid may be for L-amino acid production in microorganisms, increase L-amino acid production ability, and/or impart L-amino acid production capability.
[53]
Another example is a method of increasing the L-amino acid production capacity of the microorganism or the microorganism comprising the step of introducing (transformation) a cytochrome C encoding gene or a recombinant vector containing the gene into the microorganism. provide a way
[54]
The cytochrome C, the gene encoding it, and the microorganism are the same as described above.
[55]
As used herein, the term "vector" refers to a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and/or a sequence regulating the termination of transcription and/or translation. there is. After transformation into an appropriate host microorganism, the vector may be expressed independently of the genome (genome) of the host microorganism, or may be integrated into the genome of the host microorganism.
[56]
The vector usable in the present specification is not particularly limited as long as it is capable of replication in a host cell, and may be selected from all commonly used vectors. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, bacteriophages, and the like. For example, as the vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as a phage vector or a cosmid vector, and pBR-based, pUC as a plasmid vector system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like may be exemplified, but is not limited thereto.
[57]
The vector usable herein may be a known expression vector and/or a vector for insertion of a polynucleotide into a host cell chromosome. The insertion of the polynucleotide into the host cell chromosome may be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. The vector may further include a selection marker for confirming whether or not it is inserted into the chromosome. The selection marker is used to select cells transformed with the vector, that is, to determine whether the polynucleotide is inserted, and selectable phenotypes such as drug resistance, auxotrophic requirements, resistance to cytotoxic agents or expression of surface proteins It can be selected from among genes that confer In an environment treated with a selective agent, only cells expressing a selectable marker survive or exhibit other expression traits, and thus transformed cells can be selected.
[58]
Another example provides a method for producing L-amino acids, comprising the step of culturing the above-described L-amino acid-producing microorganisms in a medium. The method, after the step of culturing, may further include the step of recovering L-amino acids from the cultured microorganism, the medium, or both.
[59]
　In the method, the step of culturing the microorganism is not particularly limited thereto, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, and the like. At this time, the culture conditions are not particularly limited thereto, but use a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid) to an appropriate pH (eg, pH 5 to 9, specifically can control pH 6 to 8, most specifically pH 6.8) and maintain aerobic conditions by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20 to 45 °C, or 25 to 40 °C, and may be cultured for about 10 to 160 hours, but is not limited thereto. L-amino acids (eg, L-lysine) produced by the culture may be secreted into the medium or remain in cells.
[60]
The medium usable for the culture includes sugars and carbohydrates (eg, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (eg, soybean oil, sunflower oil, Peanut oil and coconut oil), fatty acids (eg palmitic acid, stearic acid and linoleic acid), alcohols (eg glycerol and ethanol), organic acids (eg acetic acid), etc. are individually used or Alternatively, two or more types may be mixed and used, but the present invention is not limited thereto. Nitrogen sources include nitrogen-containing organic compounds (e.g. peptone, yeast extract, broth, malt extract, corn steep liquor, soy meal and urea), inorganic compounds (e.g. ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and nitric acid) ammonium), etc., may be used individually or in mixture of two or more selected from the group consisting of, but is not limited thereto. As a phosphorus source, at least one selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a corresponding sodium-containing salt may be used individually or in combination of two or more, but is not limited thereto. In addition, the medium may contain essential growth-promoting substances such as other metal salts (eg, magnesium sulfate or iron sulfate), amino acids, and/or vitamins.
[61]
The step of recovering the L-amino acid (eg, L-lysine) may be collecting the desired amino acid from the medium, the culture medium, or the microorganism using a suitable method known in the art according to the culture method. For example, the recovering may be performed by one or more methods selected from centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, and the like. The method for recovering the L-amino acid (eg, L-lysine) may further include a purification step before, simultaneously with, or after that.
[62]
Effects of the Invention
[63]
By introducing a foreign gene, the lysine-producing activity of the lysine-producing strain is enhanced and maintained until the latter half of the culture, it is possible to improve the lysine-producing ability.
[64]
Brief description of the drawing
[65]
1 is a schematic diagram showing a nucleic acid sequence analysis result of a library vector obtained in Example.
[66]
Modes for carrying out the invention
[67]
Hereinafter, the present invention will be described in more detail by way of examples, but this is merely exemplary and is not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below can be modified without departing from the essential gist of the invention.
[68]
[69]
Example 1: Construction of vector library for gene introduction
[70]
In order to search for genes effective for improving the lysine production ability of Corynebacterium glutamicum , a genomic DNA library derived from extremophile bacteria that adapts and lives in various extreme environments was prepared. As extreme microorganisms, 4 representative microorganisms that can grow under extreme conditions of high osmotic pressure, high temperature, low oxygen, and various hydrogen ion concentrations, namely, Bacillus atrophaeus (ATCC 49337), Bacillus licheniformis (KCTC 1030), Lactobacillus fermentum (KCTC 3112), and Bacillus pseudofirmus OF4 (ATCC BAA2126) was used.
[71]
First, genomic DNA was obtained from the four strains using the QIAamp DNA Micro Kit (QIAGEN). The obtained genomic DNA is treated with restriction enzyme Sau3A1 (NEB), reacted at 37°C for 10 minutes, and then reacted at 65°C for 30 minutes to make an incomplete gene fragment, which is electrophoresed on a 1% agarose gel to produce a 5 to 7kb gene fragment. only acquired. For the obtained gene fragment, the gene fragment for insertion was obtained using the GeneAll Expin GEL SV kit (seoul, KOREA).
[72]
The thus-obtained gene fragment is linked to the pECCG117 vector (Korean Patent No. 0057684) obtained after a reaction with restriction enzyme BamHI-HF (NEB) at 37°C for 1 hour and CIP (NEB) enzyme treatment at 37°C for 30 minutes, E. coli DH5α was transformed and plated on LB solid medium containing kanamycin (25 mg/l). Transformed colonies were subjected to PCR using the primers of SEQ ID NOs: 1 and 2 in Table 1 below. PCR conditions were denatured at 95°C for 10 minutes, followed by denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 4 minutes repeated 30 times, followed by polymerization at 72°C for 10 minutes.
[73]
[Table 1]
Description sequence (5' -> 3') SEQ ID NO:
F primer TAA TAC GAC TCA CTA TAG GG 1
R primer CAA TTA ACC CTC ACT AAA 2
[74]
It was confirmed that a 3 to 5 kb genomic DNA fragment derived from extremophile bacteria was inserted through the PCR-amplified gene fragment, and the gene insertion rate in the pECCG117 vector was 99% or more. 10000 colonies thus transformed were obtained per strain, plasmids were extracted using the plasmid prep kit (QIAGEN), and each of these library vectors was converted into p117-Lib.Bat ( derived from Bacillus atrophaeus ), p117-Lib.Bli ( Bacillus licheniformis ), p117-Lib.Lfe ( derived from Lactobacillus fermentum ), and p117-Lib.Bps ( derived from Bacillus pseudofirmus OF4), respectively.
[75]
[76]
Example 2: Construction and screening of strains introduced into the vector library
[77]
The four library vectors (p117-Lib.Bat, p117-Lib.Bli, p117-Lib.Lfe, and p117-Lib.Bps) prepared in Example 1 were used to produce lysine, Corynebacterium glutamicum KCCM11016P. By transforming the strain (Korean Patent Registration No. 10-0159812) with an electric pulse method (Van der Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999), kanamycin (25 mg/l) was It was smeared on the included complex plate medium. Finally, about 5000 colonies were obtained for each library vector, and these were named LYS_Lib.Bat, LYS_Lib.Bli, LYS_Lib.Lfe, and LYS_Lib.Bps libraries, respectively. As a control of the library strains, the KCCM11016P strain was transformed with the pECCG117 vector into which the gDNA-derived gene fragment was not inserted, and was named LYS_117 control.
[78]
The composition of the complex plate medium used was as follows:
[79]

[80]
Glucose 10 g, Peptone 10 g, Beef extract 5 g, yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, urea 2 g, Sorbitiol 91 g, agar 20 g (based on 1 liter of distilled water)
[81]
[82]
The four KCCM11016P-based library strains obtained above were inoculated using a colony-picker (SINGER, PIXL) on a 96-Deep Well Plate-Dome (Bioneer) containing 400ul screening medium, respectively, and in a plate shaking incubator (TAITEC). Incubated for 15 hr at 32 ℃, 12000 rpm conditions.
[83]
The composition of the seed medium used was as follows:
[84]

[85]
Glucose 45 g, sugar beet-derived molasses 10 g, soybean steep liquor 10 g, (NH4)2SO4 24 g, MgSO47H2O 0.6 g, KH2PO4 0.55 g, urea 5.5 g, biotin 0.9 mg, thiamine HCl 4.5 mg, calcium-pantothenic acid 4.5 mg, Nicotinamide 30 mg, MnSO4·5H2O 9 mg, ZnSO4·5H2O 0.45 mg, CuSO4·5H2O 0.45 mg, FeSO4·5H2O 9 mg, kanamycin 25 mg (based on 1 liter of distilled water)
[86]
[87]
Cell growth during culture was monitored using a microplate-reader (BioTek), and the concentration of glucose in the culture medium and the concentration of lysine produced were measured using a glucose analyzer (YSI) and HPLC (Shimadzu), respectively. and measured.
[88]
Through the above experiment, three strains having excellent cell growth compared to the control group and a fast glucose consumption rate were finally selected (Table 2). The three selected strains were strains into which the LYS_Lib.Bps library vector was inserted, and the yield and OD values ​​were similar compared to the control strain, LYS_117 control, but the rate of consumption per hour (g/hr) of the sampling point section was higher than 100% of the LYS_117 control. It was confirmed that the productivity was excellent as it increased to a level of 118%.
[89]
[Table 2]
strain FROM 600 Relative consumption rate 36hr Lysine Yield
12hr 36hr (%) (%)
LYS_117 control 17.1 62.7 100 15.8
LYS_Lib.Bps #257 17.4 63.5 117.4 15.9
LYS_Lib.Bps #881 16.8 64.1 111.1 15.6
LYS_Lib.Bps #4213 18.1 63.8 118.0 15.8
[90]
[91]
Example 3: gDNA library sequencing analysis
[92]
In order to confirm the sequence of the gene inserted into the three colonies selected in Example 2 LYS_Lib.Bps #257, #881, and #4213, the primers of SEQ ID NOs 1 and 2 described in Table 1 of Example 1 were used. Thus, the gDNA library gene fragment possessed by the colony was amplified by PCR. PCR conditions were the same as in Example 1, and the amplified DNA fragment was obtained using the GeneAll Expin GEL SV kit (seoul, KOREA) and nucleotide sequence analysis was performed. The analysis result confirmed the genetic information through BLAST (NCBI reference sequence NC_013791.2).
[93]
The obtained analysis result is shown in FIG. 1 . As shown in FIG. 1 , as a result of gene sequencing, LYS_Lib.Bps gene fragments of 4794bp in colony #257, 3985bp in colony #881, and 4483bp in colony #4213 were included. The complete gene ORFs commonly included in the three colonies were identified as BpOF4_13735 and BpOF4_13740, and then, additional experiments were performed on the influence of the two genes.
[94]
[95]
Example 4: Individual gene transfer vector construction and strain construction
[96]
In order to confirm the influence of individual genes on the two genes identified in Example 3, a vector for genome insertion was prepared. First, a pDZ_Δ2284 vector was prepared targeting the Ncgl2284 gene, one of the transposase, to prepare a base vector for gene insertion.
[97]
Specifically, ATCC13032 gDNA was used as a DNA template for PCR, and primers were prepared with reference to the NCBI base sequence (NC_003450.3). After denaturation at 95°C for 10 minutes with the primers of SEQ ID NOs: 3 and 4, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 1 minute were repeated 30 times, followed by polymerization at 72°C for 10 minutes. PCR was performed to obtain a 5' DNA fragment of about 900 bp. Similarly, PCR was performed under the same conditions as above using primers SEQ ID NOs: 5 and 6, and the 3' DNA fragment was amplified. The two amplified DNA fragments were purified using the GeneAll Expin GEL SV kit (seoul, KOREA), treated with restriction enzyme XbaI (NEB), and then heat-treated at 65° C. for 20 minutes (Korea Patent No. 2009-0094433) vector and E. coli DH5α were transformed after ligation using the Infusion Cloning Kit. The strain was plated on LB solid medium containing kanamycin (25 mg/l), and the nucleotide sequence of the gene inserted into the pDZ vector was confirmed to finally prepare a pDZ_Δ2284 vector.
[98]
For further enhancement (expression) vectors of individual genes, the vector, promoter, and each gene DNA fragment obtained after purification by treating the base vector pDZ_Δ2284 with restriction enzymes NdeI and CIP (NEB) and heat treatment at 65° C. for 20 minutes are linked with the Infusion Cloning Kit was produced. The promoter for the additional expression of the gene used the gapA gene promoter of SEQ ID NO: 13, and to obtain it, PCR amplification was performed using ATCC13032 gDNA (NC_003450.3) as a template and primers SEQ ID NOs: 7 and 8. PCR was performed using pfu polymerase, followed by denaturation at 95°C for 10 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 1 minute repeated 30 times, followed by polymerization at 72°C for 10 minutes. proceeded. DNA fragments of two genes, BpOF4_13735 and BPOF4_13740, were performed in the same manner as in promoter PCR. For the BpOF4_13735 gene (SEQ ID NO: 14), primers of SEQ ID NOs: 9 and 10 were used, and for BpOF4_13740 (SEQ ID NO: 15), SEQ ID NO: 11 and 12 Bacillus pseudofirmus using primers The gene was amplified from OF4 gDNA. The DNA fragments thus obtained were purified using the GeneAll Expin GEL SV kit (seoul, KOREA) and ligated with the pDZ_Δ2284 vector to prepare two types of vectors. Finally, pDZ_Δ2284::PgapA BpOF4_13735 vector and pDZ_Δ2284::PgapA BpOF4_13740 vector were constructed.
[99]
The two types of vectors prepared above were transformed by an electric pulse method into a Corynebacterium glutamicum KCCM11016P strain (Korean Patent No. 10-0159812) producing lysine, and each gene was passed through a secondary DNA-crossover. A strain fortified was prepared. The two strains thus prepared were named KCCM11016P_Δ2284::PgapA BpOF4_13735 and KCCM11016P_Δ2284::PgapA BpOF4_13740.
[100]
The used primers, promoters, nucleic acid sequences of the BpOF4 gene, and amino acid sequences encoded by the genes are summarized in Table 3 below:
[101]
[Table 3]
Description sequence (5' → 3' or N → C) SEQ ID NO:
F primer for ATCC13032 gDNA GTACCCGGGGATCCTCTAGAATCGCAATGATAGCCCATTC 3
R primer for ATCC13032 gDNA TTGGTCAAACCTCCCCTcatatgCAGAAATCCACATCAAT 4
F primer for ATCC13032 gDNA ATTGATGTGGATTTCTGcatatgAGGGGAGGTTTGACCAA 5
R primer for ATCC13032 gDNA GCCTGCAGGTCGACTCTAGAATGCATCTCTGGATGATGTG 6
F primer for gapA promoter ATTGATGTGGATTTCTGcatAAGCCTAAAAACGACCGAGC 7
R primer for gapA promoter GTTGTGTCTCCTCTAAAGATTGTAG 8
F primer for BpOF4_13735 ATCTTTAGAGGAGACACAACATGGATGAAAAAAAAAAAAGC 9
R primer for BpOF4_13735 TTGGTCAAACCTCCCCTcatTTAACGCCCCAGCCAAAAAATTCC 10
F primer for BpOF4_13740 ATCTTTAGAGGAGACACAACATGAAAGGAAGACCACTTTT 11
R primer for BpOF4_13740 TTGGTCAAACCTCCCCTcatTTATTCTGAAATAGATAGTA 12
gapA promoter AAGCCTAAAAACGACCGAGCCTATTGGGATTACCATTGAAGCCAGTGTGAGTTGCATCACATTGGCTTCAAATCTGAGACTTTAATTTGTGGATTCACGGGGGTGTAATGTAGTTCATAATTAACCCCATTCGGGGGAGCAGATCGTAGTGCGAACGATTTCAGGTTCGTTCCCTGCAAAAACTATTTAGCGCAAGTGTTGGAAATGCCCCCGTTTGGGGTCAATGTCCATTTTTGAATGTGTCTGTATGATTTTGCATCTGCTGCGAAATCTTTGTTTCCCCGCTAAAGTTGAGGACAGGTTGACACGGAGTTGACTCGACGAATTATCCAATGTGAGTAGGTTTGGTGCGTGAGTTGGAAAAATTCGCCATACTCGCCCTTGGGTTCTGTCAGCTCAAGAATTCTTGAGTGACCGATGCTCTGATTGACCTAACTGCTTGACACATTGCATTTCCTACAATCTTTAGAGGAGACACAAC 13
BpOF4_13735 Nucleic Acid Sequence ATGGATGAAAAAAGAAAAGCGATTATTATAAATGAAATTAAGTACTGGCGCGAATCAAAGCTGCTTCCCTCCCAGTATTGTGATTTCTTATTAACGCTTTATTCAGAAGGAGAGGACCTAGAGACAGCCGACTCAGGAAAGCGCTTCCGAAACATTCGGACAATCTATTCGTTTATTATTGTTCAGCTTTCATTTGTCTTTACTGCTCTTGTCATTTATTTTACTGATTTTTCAAATGGATTGCAAATGCTTATTGGTTTGACTTTTTCGATTATTGTGTTAATTATAGCAAAACGGACTAGGGCAGATGCCTTTTTTCTTAAACAATTTTACTATTTTATAGGGGCTCTGATCCTCTTTTTACTAACGATTGAATGGGTTGTTCACTACAAAAGTACTAATAACCTTTTATTATCAGCAACAATCATTTTACATTGCGTTTTTTGGCTCTTTGCAGGGCTGAAATGGAAAATGCGATTTTTTACGATATCTGCTATACTAGGACTAGTAGTGTTAGGAATTTTTTGGCTGGGGCGTTAA 14
BpOF4_13740 Nucleic Acid Sequence ATGAAAGGAAGACCACTTTTACCATTTGCGATCATAGCAATTGTCGGGATTGTTGTTATGATTTCGCTTTCATTTATTGGGTTAAACCAGCGTGAAGCGATGCAGGCAGATGAAGAAGGAGAAGAAGAAGTAACTGAAATTGAAGATCCGGTAGCAGCTGGAGAAGAATTAGTGCAAACTTCTTGTATCGGTTGTCACGGTGGCGATTTAAGCGGTGGTGCAGGTCCTGCCCTAACGTCTCTTGAAGGTCAATACACTCAAGAAGAAATTACAGATATTGTTGTTAATGGGATTGGATCAATGCCGTCAGTTAACGATAACGAAGTAGAAGCAGACGCAATTGCACAGTATTTACTATCTATTTCAGAATAA 15
BpOF4_13740 amino acid sequence MKGRPLLPFAIIAIVGIVVMISLSFIGLNQREAMQADEEGEEEVTEIEDPVAAGEELVQTSCIGCHGGDLSGGAGPALTSLEGQYTQEEITDIVVNGIGSMPSVNDNEVEADAIAQYLLSISE* 16
[102]
[103]
Example 5: Analysis of lysine - producing ability of individual gene insertion strains
[104]
The two strains prepared in Example 4 were cultured in the following manner to measure OD, lysine production yield, and sugar consumption rate (g/hr). First, each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of a seed medium, and cultured with shaking at 30° C. for 20 hours at 150 rpm. Then, a 250 ml corner-baffle flask containing 24 ml of the production medium was inoculated with 1 ml of the seed culture and cultured with shaking at 32° C. for 40 hours at 150 rpm. The composition of the species medium and the production medium is as follows, respectively, and the culture results are shown in Table 4.
[105]

[106]
Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO47H2O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 2000 μg (distilled water 1) liter)
[107]

[108]
Glucose 45 g, sugar beet-derived molasses 10 g, soybean steep liquor 10 g, (NH4)2SO4 24 g, MgSO47H2O 0.6 g, KH2PO4 0.55 g, urea 5.5 g, CaCO3 30 g, biotin 0.9 mg, thiamine HCl 4.5 mg, calcium- Pantothenic acid 4.5 mg, nicotinamide 30 mg, MnSO4·5H2O 9 mg, ZnSO4·5H2O 0.45 mg, CuSO4·5H2O 0.45 mg, FeSO4·5H2O 9 mg, kanamycin 25 mg (based on 1 liter of distilled water)
[109]
[Table 4] Measurement of individual gene-enhanced strain OD, lysine production capacity, and sugar consumption rate
strain FROM Relative
consumption rate FN (end point) lysine yield
FN (%) (%)
KCCM11016P 68.5 99.5 18.9
KCCM11016P_Δ2284 67.8 100 18.7
KCCM11016P_Δ2284::PgapA BpOF4_13735 68.3 103 18.8
KCCM11016P_Δ2284::PgapA BpOF4_13740 69.1 131.5 18.6
[110]
Among the two genes obtained through the gDNA library, the strain overexpressing BpOF4_13735 did not show significant improvement in lysine yield and sugar consumption rate compared to the parent strain KCCM11016P_Δ2284. However, the KCCM11016P_Δ2284::PgapA BpOF4_13740 strain had similar fermentation final OD and yield compared to the parent strain, but it was confirmed that the rate of sugar consumption per hour in the middle section of the culture (17 to 24 hours) increased by 31.5% compared to the parent strain KCCM11016P_Δ2284.
[111]
Finally, it was confirmed that the effect of the colony LYS_Lib.Bps #257, #881, and #4213 strains confirmed in Example 2 was the effect of strengthening BpOF4_13740.
[112]
[113]
Example 6: Improvement of lysine-producing ability of the BpOF4_13740 gene-enhanced strain
[114]
In order to secondarily verify the effect of the BpOF4_13740 gene identified in Example 5, the BpOF4_13740 gene was analyzed after gene enhancement with another promoter. In addition, the effect was also confirmed for the BpOF4_05495 ​​gene, which was additionally confirmed by NCBI BLAST analysis.
[115]
For this purpose, a gene expression vector was additionally prepared. A vector was prepared in the same manner as the vector prepared in Example 3 above:
[116]
The sigB promoter was amplified by PCR using ATCC13032 gDNA as a template and primers of SEQ ID NOs: 17 and 18, and BpOF4_13740 gene was PCR amplified using the primers of SEQ ID NOs: 19 and 12 using Bacillus pseudofirmus OF4 gDNA as a template. These two gene fragments were ligated with the pDZ_Δ2284 vector to further construct a pDZ_Δ2284::PsigB BpOF4_13740 vector.
[117]
BpOF4_05495 ​​gene enhancement was performed in the same manner to construct pDZ_Δ2284::PsigB BpOF4_05495 ​​and pDZ_Δ2284::PgapA BpOF4_05495 ​​vectors. The BpOF4_05495 ​​gene fragment was obtained using SEQ ID NOs: 20 and 21 and SEQ ID NOs: 22 and 21 as primers. Meanwhile, in order to confirm the simultaneous enhancement effect of these two genes, SEQ ID NOs: 23 and 24 primers into which ribosome attachment sequences (RBS) were inserted were added, and pDZ_Δ2284::PsigB BpOF4_13740_05495 ​​and pDZ_Δ2284::PgapA BpOF4_13740_05495 ​​vectors were also constructed.
[118]
The five additional vectors constructed above (pDZ_Δ2284::PsigB BpOF4_13740 vector, pDZ_Δ2284::PsigB BpOF4_05495 ​​vector, pDZ_Δ2284::PgapA BpOF4_05495 ​​vector, pDZ_Δ2284::PsigB BpOF4_13740_pOF4_13740_05495 ​​vector producing lysine vector, pDZ_Δ2284::PsigB BpOF4_13740_05495 ​​vector) Corynebacterium glutamicum KCCM11016P strain (Korean Patent No. 10-0159812) was transformed by an electric pulse method, and strains in which each gene was enhanced through secondary DNA-crossover were prepared.
[119]
The five strains additionally prepared in this way were named KCCM11016P_Δ2284::PsigB BpOF4_13740, KCCM11016P_Δ2284::PsigB BpOF4_05495, KCCM11016P_Δ2284::PgapA BpOF4_05495, KCCM11016P_Δ2284::Psig_05495, KCCM11016P_Δ2284::Psig_p
[120]
The used primers, promoters, nucleic acid sequences of the BpOF4_05495 ​​gene and amino acid sequences encoded by the genes are summarized in Table 5 below:
[121]
[Table 5]
Description sequence (5' → 3' or N → C) SEQ ID NO:
F primer for sigB promoter ATTGATGTGGATTTCTGcatTGCAGCACCTGGTGAGGTGG 17
R primer for sigB promoter AACTGGCCTCCTAAATTCGCGGTTC 18
F primer for BPOF4_13740 GCGAATTTAGGAGGCCAGTTATGAAAGGAAGACCACTTTT 19
F primer for BpOF4_05495 GCGAATTTAGGAGGCCAGTTATGAAAAAAGTTTTTTATTAGC 20
R primer for BpOF4_05495 TTGGTCAAACCTCCCCTcatTTATTGAGCTTCAAGCCATG 21
F primer for BpOF4_05495 ATCTTTAGAGGAGACACAACATGAAAAAAGTTTTTTATTAGC 22
R primer for RBS insertion CTGTGTTTCCTCCTTTCTCCTGTTATTCTGAAATAGATAGTA 23
F primer for RBS insertion CAGGAGAAAGGAGGAAACACAGATGAAAAAGTTTTATTAGC 24
sigB promoter TGCAGCACCTGGTGAGGTGGCTGAGCCGGTGATTGAAAAGATTGCACAAGGTTTACGTGAGCGCGGAATCACCGTGGAACAAGGACGATTCGGCGCAATGATGAAGGTCACATCGGTTAACGAAGGCCCCTTCACCGTTTTGGTCGAGTGCTAGCCAGTCAATCCTAAGAGCTTGAAACGCCCCAATGTGGGGGTGTTAAGAACTCCATAAAAGCGCTTGGGAACTTTTTGTGGAAGCAGTCCGTTGAACCTCTTGAACCGCGAATTTAGGAGGCCAGTT 25
BPOF4_05495 ​​Nucleic Acid Sequence ATGAAAAAGTTTTTATTAGCTCTTGGCGCAGTTGTTGCTCTTACAGCATGTGGCGGCGGAGACGAAGCTGCTCCACCGGTTGATGAGGAGTCTCCAGCAGTAGATGAAGCTCCAGCAGATGAGCCTGCAGATGATGCAACAGCTGGTGATTACGATGCAGAATCAGCTCGTGCTACATATGAGCAAAGCTGTATCGCATGTCATGGCGGCGATCTTCAAGGGGCATCAGGTCCAGCTCTAGTAGGAACTGGCCTGTCAGCTGCTGAAATTCAAGACATCATCCAAAACGGACAAGGTTCAATGCCTGCTCAAAATTTAGATGATGACGAAGCTGCTAACCTAGCTGCATGGCTTGAAGCTCAATAA 26
BPOF4_05495 ​​amino acid sequence MKKFLLALGAVVALTACGGGDEAAPPVDEESPAVDEAPADEPADDATAGDYDAESARATYEQSCIACHGGDLQGASGPALVGTGLSAAEIQDIIQNGQGSMPAQNLDDDEAANLAAWLEAQ 27
[122]
The five strains prepared above, KCCM11016P_Δ2284::PsigB BpOF4_13740, KCCM11016P_Δ2284::PsigB BpOF4_05495, KCCM11016P_Δ2284::PgapA BpOF4_05495, KCCM11016P_ΔP_2284::PsigB5495 BpOF_4 with the same conditions as those of Example: KCCM11016P_ΔP_A2284::PsigB BapOF: In the process, the results are shown in Table 6.
[123]
[Table 6] Measurement of individual and combination gene-enhanced strain OD, lysine production capacity, and sugar consumption rate
strain FROM FN Lysine Concentration FN Lysine Yield Relative
consumption rate
FN (g/L) % (%)
KCCM11016P 68.4 9.0 18.4 101.6
KCCM11016P_Δ2284 68.1 8.7 18.7 100
KCCM11016P_Δ2284::PsigB BpOF4_13740 67.5 8.4 18.1 107.1
KCCM11016P_Δ2284::PgapA BpOF4_13740 68.8 9.2 18.4 142.1
KCCM11016P_Δ2284::PsigB BpOF4_05495 68.8 8.7 18.2 120.2
KCCM11016P_Δ2284::PgapA BpOF4_05495 68.9 8.9 17.8 136.6
KCCM11016P_Δ2284::PsigB BpOF4_13740_05495 68.4 9.3 19.3 114.8
KCCM11016P_Δ2284::PgapA BpOF4_13740_05495 69 9.5 19.0 145.9
[124]
When the BpOF4_13740 gene was expressed with the sigB promoter and the gapA promoter, it was confirmed that the rate of glucose consumption per hour increased by 7.1% and 42.1%, respectively, compared to the control KCCM11016P_Δ2284. In addition, when the BpOF4_05495 ​​gene, a similar protein, was additionally introduced into the sigB and gapA promoters, the rate of glucose consumption increased by 20.2% and 36.6%, respectively. Through the above two results, it was confirmed that the rate of sugar consumption (g/hr) increased according to the degree of gene enhancement according to the promoter strength. Additionally, the highest rate of sugar consumption was confirmed when the co-expression of the genes BpOF4_13740 and BpOF4_05495 ​​was enhanced. Specifically, the hourly glucose consumption rate of the KCCM11016P_Δ2284::PgapA BpOF4_13740_05495 ​​strain was improved by 45.9% compared to the control KCCM11016P_Δ2284.
[125]
The KCCM11016P_Δ2284::PgapA BpOF4_13740_05495 ​​strain ( named Corynebacterium glutamicum CM03-885), which showed an increase in lysine production capacity in this example, was deposited at the Korea Microorganism Conservation Center located in Hongje-dong, Seodaemun-gu, Seoul, Korea on December 13, 2019, and was assigned a deposit number.
[126]
[127]
Example 7: Analysis of lysine production capacity of the BpOF4_13740_05495 ​​enhanced strain
[128]
The genes selected in Example 6 were enhanced in Corynebacterium glutamicum KCCM10770P (Korean Patent No. 10-0924065) and KCCM11347P (Korean Patent No. 10-0073610) that produce L-lysine, respectively. As a strengthening method, additional gene introduction was carried out in the same manner as in Example 6, and finally, three vectors, pDZ_Δ2284, pDZ_Δ2284::PsigB BpOF4_13740_05495, and pDZ_Δ2284::PgapA BpOF4_13740_05495, were electropulsed with Corynebacter. 리움 글루타미쿰 KCCM10770P 및 KCCM11347P 균주 2종에 각각 도입하여, KCCM10770P_Δ2284, KCCM10770P_Δ2284::PsigB BpOF4_13740_05495, KCCM10770P_Δ2284::PgapA BpOF4_13740_05495, KCCM11347P_Δ2284, KCCM11347P_Δ2284::PsigB BpOF4_13740_05495, 및 KCCM11347P_Δ2284::PgapA BpOF4_13740_05495의 6종의 균주를 제작 did.
[129]
The prepared gene-enhanced strains were cultured in the same manner as in Example 5, and OD, lysine production yield, and relative glucose consumption rate per hour (based on 100% of the rate of glucose consumption per hour of KCCM10770P_Δ2284 and KCCM11347P_Δ2284, respectively) were measured, and the results are as follows. Table 7 shows.
[130]
[Table 7] Measurement of gene-enhanced strain OD, lysine production capacity, and relative consumption rate
strain FROM FN Lysine Concentration FN Lysine Yield Relative Speed ​​Consumed
FN (g/L) (%) (%)
KCCM10770P 95.5 6.7 13.3 99.4
KCCM10770P_Δ2284 95.3 6.5 13.0 100
KCCM10770P_Δ2284::PsigB BpOF4_13740_05495 95.0 6.5 13.0 102.5
KCCM10770P_Δ2284::PgapA BpOF4_13740_05495 95.9 6.3 12.7 105.6
KCCM11347P 65.0 15.1 30.2 99.4
KCCM11347P_Δ2284 64.7 15.3 30.6 100
KCCM11347P_Δ2284::PsigB BpOF4_13740_05495 65.5 15.1 30.2 103.5
KCCM11347P_Δ2284::PgapA BpOF4_13740_05495 65.2 15.5 31.0 108.2
[131]
As shown in Table 7, although the OD, FN lysine concentration and lysine production yield level were similar in the gene-enhanced strain prepared in this Example, the effect of shortening the fermentation time due to the improvement of the sugar consumption rate was confirmed.
[132]
[133]
Example 8: Production of CJ3P strain introduced with BpOF4_13740_05495 ​​and analysis of lysine production capacity
[134]
In order to confirm whether the same effect as above also in other strains belonging to Corynebacterium glutamicum producing L-lysine, three mutations [pyc(P458S), hom(V59A), lysC(T311I) in the wild strain )] was introduced to Corynebacterium glutamicum CJ3P (Binder et al. Genome Biology 2012, 13:R40) having L-lysine-producing ability, the BpOF4_13740_05495 ​​enhanced strain in the same manner as in Example 7 was produced. The prepared strains were named CJ3_Δ2284, CJ3_Δ2284::PsigB BpOF4_13740_05495, and CJ3_Δ2284::PgapA BpOF4_13740_05495, respectively. The control CJ3P strain (BpOF4_13740_05495 ​​enhancement was not performed) and three production strains were cultured in the same manner as in Example 5 above, and OD, lysine production yield, and relative glucose consumption rate per hour (the rate of consumption per hour of KCCM10770P_Δ2284 and KCCM11347P_Δ2284, respectively) were cultured in the same manner as in Example 5. 100% standard) was measured, and the results are shown in Table 8 below:
[135]
[Table 8] Measurement of gene-enhanced strain OD, lysine production capacity, and relative consumption rate
strain FROM FN Lysine Concentration FN Lysine Yield Relative consumption rate
FN (g/L) (%) (%)
CJ3 70.5 4.5 9.0 100.4
CJ3_Δ2284 71.4 4.2 8.4 100
CJ3_Δ2284::PsigB BpOF4_13740_05495 70.9 4.4 8.8 102.9
CJ3_Δ2284::PgapA BpOF4_13740_05495 71.6 4.6 9.2 160.9
[136]
As shown in Table 8, although the OD value, FN lysine concentration, and lysine production yield level were similar in the strain enriched with BpOF4_13740_05495, the effect of improving the sugar consumption rate per hour by more than 60% was confirmed.
[137]
[138]
Above, each description and embodiment disclosed in this specification may be applied to each other description and embodiment. All possible combinations of the various elements disclosed herein fall within the scope of the invention proposed herein. In addition, it cannot be said that the scope of the invention is limited by the specific descriptions described below, and as long as those of ordinary skill in the art can recognize or ascertain many equivalents to the specific embodiments described herein , such equivalents are intended to be encompassed by the invention proposed herein.
[139]
[Correction 04.01.2021 according to Rule 91]　

Claims
[Claim 1]
Bacillus ( Bacillus ) The activity of cytochrome C of 90 to 150 amino acids in length derived from microorganisms of the genus is enhanced, L- amino acid producing microorganisms.
[Claim 2]
According to claim 1, wherein the cytochrome C has an absorbance of 550 to 555 nm, L- amino acid producing microorganism.
[Claim 3]
The L-amino acid producing microorganism according to claim 1, wherein the cytochrome C is at least one selected from the group consisting of cytochrome c-551 and cytochrome c-550.
[Claim 4]
The microorganism producing L-amino acids according to claim 1, wherein the cytochrome C is cytochrome c-551 derived from Bacillus pseudofirmus OF4.
[Claim 5]
The method according to claim 4, wherein the Bacillus pseudofirmus OF4 derived cytochrome c-551 is a polypeptide comprising an amino acid sequence encoded by cccA, a polypeptide comprising an amino acid sequence encoded by cccB, or both. That comprising a, L- amino acid producing microorganisms.
[Claim 6]
According to claim 5, wherein the cccA will encode an amino acid sequence having SEQ ID NO: 16 or more than 80% homology thereto, L- amino acid producing microorganism.
[Claim 7]
According to claim 5, wherein the cccB will encode an amino acid sequence having SEQ ID NO: 27 or more than 80% homology thereto, L- amino acid producing microorganism.
[Claim 8]
The L-amino acid producing microorganism according to claim 1, wherein the rate of sugar consumption is increased compared to the homogeneous unmodified microorganism in which cytochrome C activity is not enhanced.
[Claim 9]
According to claim 1, wherein the microorganism is Corynebacterium ( Corynebacterium ) genus or Escherichia genus microorganisms, L- amino acid producing microorganisms.
[Claim 10]
The L-amino acid producing microorganism according to any one of claims 1 to 9, wherein the L-amino acid producing ability is increased as compared to the homogeneous unmodified microorganism in which cytochrome C activity is not enhanced.
[Claim 11]
11. The method of claim 10, wherein the L- amino acid is L-lysine, L- amino acid producing microorganism.
[Claim 12]
10. The method of any one of claims 1 to 9, comprising the steps of culturing the L-amino acid-producing microorganism of any one of claims 1 to 9 in a medium, and recovering the L-amino acid from the cultured microorganism, the medium, or both. production method.
[Claim 13]
The method of claim 12 , wherein the L-amino acid is L-lysine.
[Claim 14]
A gene encoding one or more cytochrome C selected from the group consisting of cytochrome c-551 and cytochrome c-550 derived from a microorganism of the genus Bacillus, a recombinant vector including the gene, or the gene or the recombinant vector comprising A composition for the production of L-amino acids, including cells.
[Claim 15]
The composition for producing L-amino acids according to claim 14, wherein the cytochrome C is cytochrome c-551 derived from Bacillus pseudofirmus OF4.
[Claim 16]
The method according to claim 15, wherein the cytochrome c-551 derived from Bacillus pseudofirmus OF4 is a polynucleotide comprising an amino acid sequence encoded by cccA, a polynucleotide comprising an amino acid sequence encoded by cccB, or both. A composition for producing L-amino acids comprising a.
[Claim 17]
The composition for producing L-amino acids according to claim 16, wherein the cccA encodes an amino acid sequence having SEQ ID NO: 16 or more than 80% homology thereto.
[Claim 18]
The composition for producing L- amino acids according to claim 16, wherein the cccA encodes an amino acid sequence having SEQ ID NO: 27 or more than 80% homology thereto.
[Claim 19]
The composition for producing L-amino acids according to claim 14, wherein the L-amino acids are produced in the microorganisms of the genus Corynebacterium or Escherichia.
[Claim 20]
The composition for producing L-amino acids according to any one of claims 14 to 19, wherein the L-amino acid is L-lysine.