KR102134375B1 - Microorganism for L-Lysine Production with Enhanced Cytochrome C Activity and Method of L-Lysine Production Using the Same - Google Patents

Abstract

Provided are a microorganism for L-amino acid production with enhanced cytochrome C activity, and a method for producing L-amino acids using the microorganism, wherein the microorganism is Corynebacterium glutamicum.

Description

Microorganism for L-Lysine Production with Enhanced Cytochrome C Activity and Method of L-Lysine Production Using the Same}

Provided is a cytochrome C (Cytochrome C) activity enhanced L-amino acid producing microorganism and a method for producing L-amino acid using the microorganism.

Microorganisms of the genus Corynebacterium are Gram-positive microorganisms that are widely used for L-amino acid production. L-amino acids, especially L-lysine, are used in the animal feed, human pharmaceutical and cosmetic industries, and are mainly produced by fermentation using Corynebacterium strains.

Many attempts have been made to improve the production method of L-amino acids using strains of the genus Corynebacterium. Among them, there is a study to improve the Corynebacterium strain producing L-amino acid by destroying or attenuating expression of a specific gene using recombinant DNA technology. In addition, studies have been conducted to amplify genes related to biosynthesis of each L-amino acid to study the effect on L-amino acid production and to improve the strain of Corynebacterium producing L-amino acid.

On the other hand, in the lysine production industry through fermentation, the production of high concentrations of lysine and the increase of lysine production capacity of microorganisms are important factors. Accordingly, efforts have been made to continuously improve microorganisms' lysine production capacity and maintain them during fermentation. However, due to various internal or external factors that inhibit the activity of microorganisms, there is a problem that it is difficult to maintain lysine production capacity until the end of culture.

Therefore, there is a need to develop a strain capable of continuously improving the production capacity of L-amino acids, for example, L-lysine, and continuously maintaining it.

One example provides a L-amino acid producing microorganism having enhanced Cytochrome C activity. For example, the enhancement of cytochrome C activity may be due to the introduction of a gene encoding cytochrome C. For example, the gene encoding the cytochrome C may be a foreign gene.

Another example provides a method for producing L-amino acid, comprising culturing the L-amino acid producing microorganism.

Another example provides a composition for producing L-amino acid or enhancing L-amino acid production in a microorganism, comprising a cytochrome C coding gene, a recombinant vector containing the gene, or both.

In an embodiment provided in the present specification, a gene is amplified for the purpose of increasing the lysine production capacity of the microorganism of the genus Corynebacterium to confirm the effect on lysine production, and on the basis of this, a strain improvement technique for amino acid production is proposed. In general, as a method for increasing the production capacity of amino acids such as lysine, a method of improving the amino acid production yield of lysine or the like possessed by a strain or a method of increasing the amount of amino acid production (productivity) such as lysine per hour. In particular, the productivity of amino acids such as lysine can be influenced by various factors such as the components of the fermentation medium, the osmotic pressure of the fermentation medium, temperature, agitation rate, and oxygen supply rate. For example, due to various physical and metabolite-existing stresses in the fermentation broth, physiological conditions such as decrease in oxygen supply due to increase in microbial cells, and physical conditions such as temperature and agitation speed, lysine production capacity of microorganisms and cell activity of microorganisms It decreases continuously. In an example of the present specification, an improvement technique of a strain is provided to overcome stress caused by various factors such as these and to maintain the production activity of a target product of microorganisms until late fermentation.

It will be described in more detail below.

An example provides an L-amino acid producing microorganism with enhanced Cytochrome C activity.

In the present specification, the term “L-amino acid producing microorganism” refers to a case where a microorganism having L-amino acid producing ability has an increased L-amino acid producing ability by enhancing cytochrome C activity and/or does not have a L-amino acid producing ability. It can be used to mean that the microorganism has the ability to produce L-amino acids by enhancing cytochrome C activity. As used herein, "microorganism" encompasses unicellular bacteria and can be used interchangeably with "cell".

The L-amino acid may be L-lysine.

In the present specification, in order to distinguish the microorganism before the cytochrome C activity is enhanced, the cytochrome C activity is enhanced, the L-amino acid production capacity is increased or the L-amino acid production capacity is assigned to the "L-amino acid production microorganism", said cytos The microorganism before chromium C activity is enhanced can be expressed as a host microorganism.

In one example, the host microorganism may be selected from all microorganisms having L-amino acid (eg, L-lysine) production capacity. In one example, the host microorganism may be a microorganism having L-lysine production capacity by introducing mutations into a microorganism having naturally L-lysine production capacity or a parent strain that has little or no L-lysine production capacity.

In one example, the host microorganism is a microorganism having natural L-lysine production capacity, or a gram-positive bacterium having L-lysine production capacity, for example, Corynebacte It may be one or more selected from the group consisting of microorganisms of the genus Corynebacterium and microorganisms of the genus Escherichia . The microorganism of the genus Corynebacterium is Corynebacterium 　 glutamicum ), Corynebacterium ammoniagenes ), Brevibacterium lactofermentum , Brevibacterium flavum , Corynebacterium thermoaminogenes ( Corynebacterium 　 thermoaminogenes ), Corynebacterium 　 efficiens ), and the like, but is not limited thereto. For example, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum .

In this specification, 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 means 100 to 125, or 120 to 125 amino acids long, cytochrome C of monomers having membrane-binding properties. In one embodiment, the cytochrome C may be derived from microorganisms of the genus Bacillus, and selected from among cytochrome C having a wavelength range of 550 to 555 nm or 550 to 551 nm in which the absorption band (absorbance) of the lowest level energy in the reduced state is exhibited. It may be one or more. 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 the microorganism of the genus Bacillus, such as one kind, It may be two types or three types (the values described after the cytochrome c above refer to absorbance). The microorganism of the genus Bacillus may be one or more selected from the group consisting of Bacillus pseudofirmus , Bacillus subtilis , and the like.

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 selected from the group consisting of Bacillus pseudofirmus (e.g., Bacillus pseudofirmus OF4) and / or a Bacillus subtilis-derived cytochrome c-551, Bacillus subtilis-derived cytochrome c-550, such as one kinds It may be abnormal.

More specifically, the cytochrome C (e.g., cytochrome c-551 from Bacillus subtilis ) is poly containing an amino acid sequence encoded by cccA (e.g., BpOF4_13740 from Bacillus pseudofirmus OF4) (e.g., SEQ ID NO: 16). It may be a peptide, a polypeptide comprising an amino acid sequence encoded by cccB (eg, BpOF4_05495 from Bacillus pseudofirmus OF4) (eg, SEQ ID NO: 27), or both.

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, and 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 more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more (e.g., 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%). As described above, the protein having the sequence homology that can be included in the cytochrome C category described in the present specification includes (1) characteristics of cytochrome C described above, such as (a) bacteria, (b) 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 acid lengths, (c) membrane binding properties, and (d) monomer properties Having one or more of, and/or (2) the effect of enhancing activity in the microorganism, such as compared to an unmodified microorganism, (e) increasing L-amino acid (eg, L-lysine) production capacity and/or (f) The effect of increasing the sugar consumption rate may be at a level equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 27 as exemplified above.

In one example, the L-amino acid-producing microorganism with enhanced cytochrome C activity may be an L-amino acid-producing ability increased as compared to a homogeneous unmodified microorganism with no enhanced cytochrome C activity.

In the present specification, the term “enhancement of cytochrome C activity” may mean any manipulation in which cytochrome C activity is introduced into a microorganism, or cytochrome C activity may be enhanced compared to the intrinsic activity or pre-modification activity of a microorganism. have. The "introduction" means that the activity of cytochrome C, which the microorganism did not originally have, appears naturally or artificially. The "unmodified microorganism" may mean a host microorganism in which cytochrome C is not enhanced or before it is enhanced. The "intrinsic activity" may refer to the activity of cytochrome C, which was originally or not enhanced by cytochrome C, which was originally possessed by the host microorganism. As used herein, "unmodified" may be used interchangeably with a form having intrinsic activity without genetic variation.

For example, enhancing cytochrome C activity can include both introducing and enhancing foreign cytochrome C, or enhancing intrinsic cytochrome C activity. In one example, the enhancement of cytochrome C activity may be to enhance the introduction of foreign cytochrome C.

In one example, the enhancement of cytochrome C activity in the microorganism can be confirmed that the sugar consumption rate of the microorganism is improved compared to the unmodified microorganism having no enhanced cytochrome C activity. In particular, the cytochrome C activity-enhanced microorganism exemplified in one embodiment has a strain growth (OD value) and/or L-amino acid, e.g., L-lysine production yield similar to that of the unmodified microorganism and a sugar consumption rate May be improved, which suggests that the cytochrome C activity-enhanced microorganism produces more L-amino acids in a shorter time than non-modified microorganisms, thereby improving L-amino acid productivity.

In one example, the enhancement of cytochrome C activity may be due to an increase in expression level at the gene (mRNA) level and/or protein level of cytochrome C and/or an increase in activity as cytochrome C, but is not limited thereto. no.

In one example, the enhancement of cytochrome C activity may be due to the introduction of a gene encoding cytochrome C. By introducing such a gene encoding cytochrome C, L-amino acid production capacity of a microorganism having L-amino acid production capacity is increased or L-amino acid production capacity can be given to a microorganism having no L-amino acid production capacity.

In one example, the cytochrome C or the gene encoding the same may be from a host microorganism (intrinsic) or from another microorganism (foreign). In one embodiment, the enhancement of cytochrome C activity may be performed by introducing polynucleotides encoding foreign cytochrome C into the host microorganism. Said cytochrome C is as described above, for example, may be cytochrome C derived from Bacillus pseudofirmus OF4 (cytochrome c-551), for example, SEQ ID NO: 16 (e.g., encoded by cccA (BpOF4_13740)) Or it may be represented by the amino acid sequence of SEQ ID NO: 27 (eg, encoded by cccB (BpOF4_05495)).

In one example, the gene encoding the cytochrome C or the L-amino acid producing microorganism in which the gene is introduced may be a polynucleotide encoding SEQ ID NO: 16, a polynucleotide encoding SEQ ID NO: 27, or both. have. In one embodiment, the L-amino acid-producing microorganism is a microorganism of the genus Corynebacterium comprising polynucleotide encoding SEQ ID NO: 16, polynucleotide encoding SEQ ID NO: 27, or both, such as Corynebacterium glue Coramibacterium glutamicum . For example, the L-amino acid-producing microorganism may be one having accession number KCCM12640P.

As used herein, a polynucleotide (which can be used interchangeably with a “gene”) or a polypeptide (which can be used interchangeably with a “protein”) comprises a “specific nucleic acid sequence or amino acid sequence, consisting of a specific nucleic acid sequence or amino acid sequence, Or “expressed as a specific nucleic acid sequence or amino acid sequence” is an interchangeable expression in an equivalent sense, and may mean that the polynucleotide or polypeptide essentially includes the specific nucleic acid sequence or amino acid sequence. Includes “substantially equivalent sequences” with variations (deletions, substitutions, modifications, and/or additions) to the particular nucleic acid sequence or amino acid sequence within a range that maintains the original and/or desired function of the nucleotide or polypeptide. It can be interpreted as (or does not exclude the introduction of the mutation).

In one example, nucleic acid sequences or amino acid sequences provided herein are conventional mutagenesis methods, such as direct evolution and/or site specificity, to the extent that they retain their original or desired function. And modified by site-directed mutagenesis. In one example, a polynucleotide or polypeptide that “comprises a specific nucleic acid sequence or amino acid sequence” means that the polynucleotide or polypeptide consists of (i) or consists essentially of the specific nucleic acid sequence or amino acid sequence, Or (ii) 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 with 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 (e.g., 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%). It may mean that the amino acid sequence has a homology or essentially contains it and maintain the original function and/or desired function. In the present specification, the original function may be a cytochrome C function (for amino acid sequence), or a function for encoding a protein having the cytochrome C function (for nucleic acid sequence), and the desired function may be a microorganism It may mean a function of increasing or imparting L-amino acid (eg, L-lysine) production capacity.

The nucleic acid sequence described herein is an amino acid sequence and/or function of a protein expressed from a coding region, considering codons preferred by microorganisms that want to express the protein (cytochrome C) due to degeneracy of the codon. Various modifications can be made to the coding region within a range that does not change.

As used herein, the term “homology” refers to the degree to which a given nucleic acid sequence or amino acid sequence matches, and may be expressed as a percentage (%). For homology to nucleic acid sequences, for example, the algorithm BLAST (see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (see Methods Enzymol) ., 183, 63, 1990). Based on this algorithm BLAST, a program called BLASTN or BLASTX has been developed (see http://www.ncbi.nlm.nih.gov).

In one example, a polynucleotide comprising a specific nucleic acid sequence provided herein comprises a polynucleotide fragment comprising the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, as well as a nucleic acid sequence complementary to the specific nucleic acid sequence. Can be interpreted as Specifically, the polynucleotide having the complementarity may be hybridized at a Tm value that can be appropriately adjusted by a person skilled in the art according to 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 the known literature. For example, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher Above, 98%, 98%, 99.5%, or 99.9% or more genes with high complementarity hybridize, and genes with lower complementarity do not hybridize, or 60, which is the washing condition for normal Southern hybridization ℃ 1x saline-sodium citrate buffer (SSC), and 0.1% (w/v) SDS (Sodium Dodecyl Sulfate); 60°C 0.1x SSC, and 0.1% SDS; Or 68 ℃ 0.1x SSC, and at a salt concentration and temperature corresponding to 0.1% SDS, the conditions for washing once, specifically 2 to 3 times, etc. may be listed, but are not limited thereto. Hybridization may require two nucleotides to have complementary sequences, or mismatches between bases may be tolerated, depending on the stringency of the hybridization. The term “complementary” can be used to describe the relationship between nucleotide bases that are hybridizable to each other. For DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. The appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide, which is well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

If the method for enhancing the cytochrome C gene (mRNA) level during the enhancement of the cytochrome C activity is described in more detail,

1) an increase in the number of copies of the polynucleotide encoding the cytochrome C,

2) modification of the expression control sequence to increase the expression of the polynucleotide, or

3) Method to be modified to be strengthened by the combination of 1) and 2) above

And the like, but is not limited thereto.

The 1) increase in the number of copies of the polynucleotide is not particularly limited, but may be performed by introducing the polynucleotide into a host microorganism through a vector or inserting into a chromosome of the host microorganism. For example, the increase in copy number can be performed by introducing a polynucleotide encoding a foreign cytochrome C or a codon optimized variant polynucleotide of the polynucleotide into a host microorganism. The foreign polynucleotide can be used without limitation in its origin or sequence as long as it exhibits the same/similar activity to the cytochrome C it encodes. The introduction may be performed by a person skilled in the art appropriately selecting and/or modifying known transformation methods. By introducing the polynucleotide introduced into the host microorganism by the expression, it is possible to generate foreign cytochrome C.

2) The modification of the expression control sequence to increase the expression of the polynucleotide is applicable to both the foreign polynucleotide and the intrinsic polynucleotide, and is not particularly limited thereto, but the expression control sequence of the nucleotide is further enhanced so that the expression control activity is further enhanced. Deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacement with expression control sequences having stronger activity. The expression control sequence is not particularly limited, but may be one or more selected from the group consisting of a promoter, an operator sequence, a sequence encoding a ribosomal binding site, a sequence controlling termination of transcription and/or translation, and the like. In one embodiment, to increase expression, a strong heterologous promoter can be operably linked to the top of the polynucleotide expression unit instead of the original promoter. Examples of the strong promoter include CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, and the like. More specifically, the strong promoter may be a lysCP1 promoter (WO2009/096689) or a CJ7 promoter (WO2006/065095), a promoter derived from the genus Corynebacterium, but is not limited thereto.

Hereinafter, a case in which the gene encoding the cytochrome C described above is introduced into the host microorganism through the vector or inserted into the chromosome of the host microorganism will be described in more detail. The introduction of the gene described herein is carried out by introducing a gene of a foreign gene (derived from a host microorganism and/or from another individual of the same kind) into a host cell in a form operably linked to a recombinant vector, or ii) a host It can be performed by inserting into a cell's chromosome (dielectric) (eg, random insertion). Wherein ii) when inserted into the chromosome (dielectric) of the host cell, the insertion site is a position that does not affect the growth of the host cell (e.g., non-transcriptional spacer (NTS), etc.) and / or random insertion efficiency It may be a position that can be increased (eg, a retrotransposon, etc.), but is not limited thereto.

The introduction of the gene or vector may be performed by appropriately selecting a known transformation method by those skilled in the art. In this specification, the term "transformation" is to introduce a vector containing a polynucleotide encoding a target protein (cytochrome C) into a host microorganism so that the protein encoded by the polynucleotide can be expressed in a host cell. it means. The transformed polynucleotide can include all of them, regardless of whether they are inserted into the chromosome of the host microorganism or located outside the chromosome, as long as it can be expressed in the host microorganism. In addition, the polynucleotide includes DNA and RNA encoding a target protein. If the polynucleotide can be expressed by being introduced into a host microorganism, the introduced form is not limited. For example, the polynucleotide may be introduced into a host microorganism in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette may include an expression control element such as a promoter, a transcription termination signal, a ribosome binding site, and/or a translation termination signal, which are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell. The term "operably linked" in the above is an expression control element (eg, a promoter) and an expression control element (eg, a promoter) so that the expression control element can perform transcription control (eg, transcription initiation) of a polynucleotide encoding a target protein (cytochrome C). It may mean that the polynucleotide is functionally linked. Operable linkages can be performed using known genetic recombination techniques in the art, such as, but not limited to, conventional site-specific DNA cleavage and linkage.

The method for transforming the polynucleotide into a host microorganism can be performed by any method in which nucleic acids are introduced into cells (microorganisms), 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 (PEG) precipitation (polyethylene glycol-mediated uptake) ), DEAE-dextran method, cationic liposome method, lipofection, lithium acetate-DMSO method, etc. may be exemplified, but is not limited thereto.

Insertion of the gene into the host cell genome (chromosome) can be performed by a person skilled in the art appropriately selecting a known method, for example, an RNA-guided endonuclease system (e.g., (a) RNA- Guide endonuclease (eg, Cas9 protein, etc.), a coding gene thereof, or a vector comprising the gene; and (b) guide RNA (eg, single guide RNA (sgRNA), etc.), coding DNA thereof, or the DNA A mixture comprising a vector comprising (e.g., a mixture of RNA-guide endonuclease protein and guide RNA, etc.), complex (e.g., ribonucleic acid fusion protein (RNP)), recombinant vector (e.g., RNA-guide endonuclease It may be performed using one or more selected from the group consisting of vectors, including vectors containing the first encoding gene and guide RNA encoding DNA, etc.), but is not limited thereto.

In another example, cytochrome C coding gene, recombinant vector comprising said gene, and/or increasing L-amino acid production capacity and/or conferring L-amino acid production capacity in microorganisms of cells containing said gene or said recombinant vector Use is provided for.

One example provides a composition for producing L-amino acid comprising a gene encoding cytochrome C, a recombinant vector containing the gene, or a cell containing the gene or the recombinant vector.

Another example may be a composition for producing L-amino acids, including a cytochrome C coding gene, a recombinant vector containing the gene, or both. The composition for producing L-amino acids may be for producing L-amino acids in microorganisms, increasing L-amino acid production capacity, and/or conferring L-amino acid production capacity.

Another example comprises the step of introducing (transforming) a cytochrome C coding gene or a recombinant vector containing the gene into a microorganism, a method for increasing the L-amino acid production capacity of the microorganism or conferring the L-amino acid production capacity to the microorganism Provides a method.

The cytochrome C, the gene encoding it, and the microorganism are as described above.

As used herein, the term "vector" refers to a DNA preparation that contains a base 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, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and/or a sequence regulating the termination of transcription and/or translation. have. The vector can be transformed into a suitable host microorganism, and then expressed independently of the host microorganism's genome (dielectric), or integrated into the host microorganism's genome.

The vector usable in this specification is not particularly limited as long as it is replicable in the host cell, and can be selected from all commonly used vectors. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophage. For example, as the vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as a phage vector or a cosmid vector, and pBR system, pUC as a plasmid vector. System, pBluescriptII system, pGEM system, pTZ system, pCL system, and pET system. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be exemplified, but are not limited thereto.

The vector usable herein may be a known expression vector and/or a vector for insertion into a host cell chromosome of a polynucleotide. The insertion of the polynucleotide into the host cell chromosome can be made 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 insertion into the chromosome. The selection marker is for selecting cells transformed with a vector, that is, to confirm whether or not the polynucleotide is inserted, and selectable phenotypes such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface proteins It can be selected and used from among the genes conferring. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit different expression traits, so that the transformed cells can be selected.

Another example provides a method for producing L-amino acid, comprising culturing the aforementioned L-amino acid-producing microbial microorganism in a medium. The method may further include, after the culturing step, recovering L-amino acid from the cultured microorganism, medium, or both.

In the above method, the step of culturing the microorganism is not particularly limited, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, or the like. In this case, the culture conditions are not particularly limited, but using a basic compound (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (for example, phosphoric acid or sulfuric acid) to an appropriate pH (eg, pH 5 to 9, specifically Can adjust 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 cultured for about 10 to 160 hours, but is not limited thereto. The L-amino acid (eg, L-lysine) produced by the above culture may be secreted into "medium" or remain in the cell.

The medium that can be used for the culture is a carbon source, sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seed oil, Peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol), organic acids (e.g. acetic acid), one or more selected from the group consisting of Or it may be used by mixing two or more, but is not limited thereto. Nitrogen sources include nitrogen-containing organic compounds (e.g. peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea), inorganic compounds (e.g. ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and nitric acid) Ammonium) may be used individually or in combination of two or more selected from the group consisting of, but is not limited thereto. As the phosphorus source, one or more selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the 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 include other metal salts (eg, magnesium sulfate or iron sulfate), amino acids, and/or essential growth-promoting materials such as vitamins.

The step of recovering the L-amino acid (eg, L-lysine) may be to collect the desired amino acid from the medium, culture medium, or microorganism using a suitable method known in the art according to the culture method. For example, the recovering step 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, or thereafter.

By introducing foreign genes, lysine-producing activity of the lysine-producing strain is enhanced and maintained until late in culture, thereby improving lysine-producing ability.

1 is a schematic diagram showing the results of nucleic acid sequence analysis of a library vector obtained in one embodiment.

Hereinafter, the present invention will be described in more detail with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that the embodiments described below can be modified without departing from the essential gist of the invention.

Example 1: Construction of vector library for gene introduction

Corynebacterium In order to search for a gene effective for improving lysine production capacity of glutamicum ), a genomic DNA library derived from extreme microorganisms (extremophile bacteria) that adapt and live in various extreme environments was constructed. Extreme microorganisms include four representative microorganisms capable of growing 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.

First, genomic DNA was obtained from the four strains using the QIAamp DNA Micro Kit (QIAGEN). The obtained genomic DNA is treated with the restriction enzyme Sau3A1 (NEB), reacts at 37°C for 10 minutes, and then reacts at 65°C for 30 minutes to make an incomplete gene fragment, and electrophoresis it on 1% agarose gel to generate a 5-7kb gene fragment. Only acquired. Gene fragments for insertion were obtained using the GeneAll Expin GEL SV kit (seoul, KOREA).

The thus obtained gene fragment is connected to the restriction enzyme BamHI-HF (NEB) at 37° C. for 1 hour, and after 30 minutes of CIP (NEB) enzyme treatment at 37° C., and the pECCG117 vector (Korea Patent No. 0057684) obtained. E. coli DH5α was transformed and streaked on LB solid medium containing kanamycin (25 mg/l). The transformed colonies were subjected to PCR using primers of SEQ ID NOs: 1 and 2 in Table 1 below. PCR conditions were denatured 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 4 minutes was repeated 30 times, followed by polymerization at 72°C for 10 minutes.

Description Sequence (5' -> 3') Sequence number F primer TAA TAC GAC TCA CTA TAG GG One R primer CAA TTA ACC CTC ACT AAA 2

Through the PCR amplified gene fragment, it was confirmed that a genomic DNA fragment derived from 3 to 5 kb of extreme microorganism (extremophile bacteria) was inserted, and the gene insertion rate in the pECCG117 vector was more than 99%. 10000 colonies thus transformed were obtained per strain, and plasmids were extracted using a plasmid prep kit (QIAGEN). Each of these library vectors was p117-Lib.Bat (from Bacillus atrophaeus ), p117-Lib.Bli ( Bacillus licheniformis ), p117-Lib.Lfe (from Lactobacillus fermentum ), and p117-Lib.Bps (from Bacillus pseudofirmus OF4).

Example 2: Vector library introduction strain production and screening

Corynebacterium glutamicum KCCM11016P producing lysine from the four library vectors prepared in Example 1 (p117-Lib.Bat, p117-Lib.Bli, p117-Lib.Lfe, and p117-Lib.Bps) The strain (Republic of Korea Patent No. 10-0159812) was transformed with an electric pulse method (Van der Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999) to give kanamycin (25 mg/l). It was smeared on the included composite plate medium. Finally, about 5000 colonies were obtained for each library vector, and these were named as LYS_Lib.Bat, LYS_Lib.Bli, LYS_Lib.Lfe, and LYS_Lib.Bps libraries, respectively. As a control of the library strains, a pECCG117 vector without gDNA-derived gene fragment inserted into the KCCM11016P strain was transformed and used, named LYS_117 control.

The composition of the composite flat plate used above is as follows:

<Composite plate medium (pH 7.0)>

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)

The obtained 4 types of KCCM11016P-based library strains were inoculated using colony-picker (SINGER, PIXL) in 96-Deep Well Plate-Dome (Bioneer) containing 400 ul screening medium, respectively, and in a plate shaking incubator (TAITEC). Incubated for 15 hr at 32°C and 12000 rpm.

The medium medium composition used is as follows:

<Screening medium (pH 7.0)>

45 g of glucose, 10 g of molasses derived from sugar beet, 10 g of soybean immersion solution, 24 g of (NH4)2SO4, 0.6 g of MgSO4.7H2O, 0.55 g of KH2PO4, urea 5.5 g, biotin 0.9 mg, thiamine HCl 4.5 mg, calcium-pantothenic acid 4.5 mg, nicotinamide 30 mg, MnSO4ㆍ5H2O 9 mg, ZnSO4ㆍ5H2O 0.45 mg, CuSO4ㆍ5H2O 0.45 mg, FeSO4ㆍ5H2O 9 mg, kanamycin 25 mg (based on 1 liter of distilled water)

Cell growth during the culture was monitored using a microplate-reader (BioTek), and the concentration of glucose present in the culture medium and the concentration of lysine produced were measured using a sugar analyzer (YSI) and an HPLC (Shimadzu) analyzer, respectively. Was measured.

Through the above experiment, finally, three strains having excellent cell growth compared to the control group and having a fast glucose consumption rate were 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 to those of the control strain LYS_117 control, but the rate of sugar consumption per hour (g/hr) of the sampling point section was 100% compared to the LYS_117 control. It was confirmed that the productivity increased by increasing to 118%.

Strain OD 600 Relative sugar 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

Example 3: gDNA Library sequencing

To confirm the sequence of the gene inserted in the three colonies selected in Example 2 LYS_Lib.Bps #257, #881, and #4213, the primers of SEQ ID NOs: 1 and 2 shown in Table 1 of Example 1 were used. Thus, the gDNA library gene fragment possessed by the colony was PCR amplified. PCR conditions were the same as in Example 1, and the amplified DNA fragment was obtained using GeneAll Expin GEL SV kit (seoul, KOREA) and sequence analysis was performed. The analysis result confirmed the genetic information through BLAST (NCBI reference sequence NC_013791.2).

The obtained analysis results are shown in FIG. 1. As shown in Figure 1, the gene sequence analysis result, LYS_Lib.Bps #257 colony contains 4794bp, #881 colony contains 3985bp, and #4213 colony contains gene fragments of 4483bp. The complete gene ORFs commonly included in the three colonies were identified as BpOF4_13735 and BpOF4_13740, and thereafter, further experiments were conducted on the influence of the two genes.

Example 4: Production of individual gene introduction vectors and production of strains

In order to confirm the individual gene impact on the two genes identified in Example 3, a vector for genome insertion was constructed. First, in order to construct a base vector for gene insertion, a pDZ_Δ2284 vector was constructed targeting the Ncgl2284 gene, one of transposases.

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 primers of SEQ ID NOS: 3 and 4, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and repeated polymerization for 1 minute at 72°C for 30 times, followed by polymerization reaction 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 amplified two DNA fragments were purified using 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 Infusion Cloning Kit, and then transformed into E. coli DH5α. The strain was spread on an LB solid medium containing kanamycin (25 mg/l) and the nucleotide sequence of the gene inserted in the pDZ vector was confirmed to finally prepare a pDZ_Δ2284 vector.

For additional reinforcement (expression) vectors of individual genes, the vector pDZ_Δ2284 is treated with the restriction enzymes NdeI and CIP (NEB) and heat-treated at 65° C. for 20 minutes to connect the vector and promoter obtained after purification and each gene DNA fragment with an Infusion Cloning Kit. Was produced. The promoter for further expression of the gene was used as the gapA gene promoter of SEQ ID NO: 13, and PCR amplification was performed using primers SEQ ID NOs: 7 and 8 as a template for ATCC13032 gDNA (NC_003450.3) to obtain this. PCR was performed using pfu polymerase, after denaturation at 95°C for 10 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and repeated polymerization for 1 minute at 72°C for 30 times, followed by polymerization at 72°C for 10 minutes. Proceeded. The two genes BpOF4_13735 and BPOF4_13740 DNA fragments were processed in the same manner as the promoter PCR, primers of SEQ ID NOs: 9 and 10 were used for the BpOF4_13735 gene (SEQ ID NO: 14), and SEQ ID NOs: 11 and 11 for the BpOF4_13740 (SEQ ID NO: 15) Genes were amplified from Bacillus pseudofirmus OF4 gDNA using primers of 12. The DNA fragments thus obtained were purified using the GeneAll Expin GEL SV kit (seoul, KOREA) to connect with the pDZ_Δ2284 vector to produce two vectors. Finally, the pDZ_Δ2284::PgapA BpOF4_13735 vector and the pDZ_Δ2284::PgapA BpOF4_13740 vector were constructed.

The two vectors prepared above were transformed by an electric pulse method into the Corynebacterium glutamicum KCCM11016P strain (Republic of Korea Patent No. 10-0159812) producing lysine, and each gene was subjected to secondary DNA-crossover. To produce the enhanced strain. The two strains thus produced were named as KCCM11016P_Δ2284::PgapA BpOF4_13735, KCCM11016P_Δ2284::PgapA BpOF4_13740.

The primers used, the promoter, the nucleic acid sequence of the BpOF4 gene, and the amino acid sequence encoded by the gene are summarized in Table 3 below:

Description Sequence (5' → 3'or N→C) Sequence number 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 ATCTTTAGAGGAGACACAACATGGATGAAAAAAGAAAAGC 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

Example 5: Individual gene insertion strain Lysine Production capacity analysis

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 seed medium, and shake cultured at 150 rpm for 20 hours at 30°C. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of production medium and shake cultured at 150 rpm for 40 hours at 32°C. The composition of the seed medium and the production medium are as follows, and the culture results are shown in Table 4.

<Vertical medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 2000 μg ( 1 liter of distilled water)

<Production medium (pH 7.0)>

45 g of glucose, 10 g of molasses derived from sugar beet, 10 g of soybean immersion solution, 24 g of (NH4)2SO4, 0.6 g of MgSO4.7H2O, 0.5 g of KH2PO4, 5.5 g of urea, 5.5 g of CaCO3, 30 g of biotin, 0.9 mg of biotin, 4.5 mg of thiamine HCl, Calcium-pantothenic acid 4.5 mg, nicotinamide 30 mg, MnSO4ㆍ5H2O 9 mg, ZnSO4ㆍ5H2O 0.45 mg, CuSO4ㆍ5H2O 0.45 mg, FeSO4ㆍ5H2O 9 mg, kanamycin 25 mg (based on 1 liter of distilled water)

Measurement of individual genetically enhanced strain OD, lysine production capacity, sugar consumption rate Strain OD opponent
Sugar consumption rate FN 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

Among the two genes obtained through the gDNA library, the strain overexpressing BpOF4_13735 did not show a significantly improved effect on lysine yield and sugar consumption rate compared to the parent strain KCCM11016P_Δ2284. However, the strains of KCCM11016P_Δ2284::PgapA BpOF4_13740 were similar to the final fermentation OD and yield compared to the parent strain, but it was confirmed that the rate of sugar consumption per hour in the intermediate section (17 to 24 hours) increased by 31.5% compared to the parent strain KCCM11016P_Δ2284.

Finally, it was confirmed that the effect of the colonies LYS_Lib.Bps #257, #881, and #4213 strains identified in Example 2 was an effect by strengthening BpOF4_13740.

Example 6: BpOF4_13740 gene-enhanced strain enhanced lysine production capacity

In order to secondaryly verify the effect of the BpOF4_13740 gene identified in Example 5, gene reinforcement was performed with another promoter, and the effect was also confirmed for the BpOF4_05495 gene further confirmed by NCBI BLAST analysis.

To this end, a gene expression vector was additionally constructed. The vector was prepared in the same process as the vector produced in Example 3 above:

PCR was amplified by using the primers of SEQ ID NOs: 17 and 18 as a template for ATCC13032 gDNA to amplify the sigB promoter, and the BpOF4_13740 gene was PCR amplified by using primers of SEQ ID NOs: 19 and 12 as a template for Bacillus pseudofirmus OF4 gDNA. The two gene fragments were linked to the pDZ_Δ2284 vector to further construct the pDZ_Δ2284::PsigB BpOF4_13740 vector.

The gene enhancement of BpOF4_05495 was also performed in the same manner to construct pDZ_Δ2284::PsigB BpOF4_05495 and pDZ_Δ2284::PgapA BpOF4_05495 vectors. The primers were obtained using the SEQ ID NOs: 20 and 21 and SEQ ID NOs: 22 and 21 to obtain the BpOF4_05495 gene fragment. On the other hand, in order to confirm the co-strengthening effect of these two genes, primers SEQ ID NOs 23 and 24 in which ribosome attachment sequences (RBS) were inserted were added to prepare pDZ_Δ2284::PsigB BpOF4_13740_05495 and pDZ_Δ2284::PgapA BpOF4_13740_05495 vectors.

The five additional vectors produced above (pDZ_Δ2284::PsigB BpOF4_13740 vector, pDZ_Δ2284::PsigB BpOF4_05495 vector, pDZ_Δ2284::PgapA BpOF4_05495 vector, pDZ_Δ2284::PsigB BpOF4_1384_P4ig: The strain of Corynebacterium glutamicum KCCM11016P (Republic of Korea Patent No. 10-0159812) was transformed by an electric pulse method, and each gene was strengthened through a secondary DNA-crossover.

Five additional strains produced as described above were KCCM11016P_Δ2284::PgapA BpOF4_13740, KCCM11016P_Δ2284::PsigB BpOF4_05495, KCCM11016P_Δ2284::PgapA BpOF4_05495, KCCM11016P_Δ2284:84P_P_Pigig

The primers, promoters, nucleic acid sequences of the BpOF4_05495 gene and amino acid sequences encoded by the genes are summarized in Table 5 below:

Description Sequence (5' → 3'or N→C) Sequence number 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 GCGAATTTAGGAGGCCAGTTATGAAAAAGTTTTTATTAGC 20 R primer for BpOF4_05495 TTGGTCAAACCTCCCCTcatTTATTGAGCTTCAAGCCATG 21 F primer for BpOF4_05495 ATCTTTAGAGGAGACACAACATGAAAAAGTTTTTATTAGC 22 R primer for RBS insertion CTGTGTTTCCTCCTTTCTCCTGTTATTCTGAAATAGATAGTA 23 F primer for RBS insertion CAGGAGAAAGGAGGAAACACAGATGAAAAAGTTTTTATTAGC 24 sigB promoter TGCAGCACCTGGTGAGGTGGCTGAGCCGGTGATTGAAAAGATTGCACAAGGTTTACGTGAGCGCGGAATCACCGTGGAACAAGGACGATTCGGCGCAATGATGAAGGTCACATCGGTTAACGAAGGCCCCTTCACCGTTTTGGTCGAGTGCTAGCCAGTCAATCCTAAGAGCTTGAAACGCCCCAATGTGGGGGTGTTAAGAACTCCATAAAAGCGCTTGGGAACTTTTTGTGGAAGCAGTCCGTTGAACCTCTTGAACCGCGAATTTAGGAGGCCAGTT 25 BPOF4_05495 nucleic acid sequence ATGAAAAAGTTTTTATTAGCTCTTGGCGCAGTTGTTGCTCTTACAGCATGTGGCGGCGGAGACGAAGCTGCTCCACCGGTTGATGAGGAGTCTCCAGCAGTAGATGAAGCTCCAGCAGATGAGCCTGCAGATGATGCAACAGCTGGTGATTACGATGCAGAATCAGCTCGTGCTACATATGAGCAAAGCTGTATCGCATGTCATGGCGGCGATCTTCAAGGGGCATCAGGTCCAGCTCTAGTAGGAACTGGCCTGTCAGCTGCTGAAATTCAAGACATCATCCAAAACGGACAAGGTTCAATGCCTGCTCAAAATTTAGATGATGACGAAGCTGCTAACCTAGCTGCATGGCTTGAAGCTCAATAA 26 BPOF4_05495 amino acid sequence MKKFLLALGAVVALTACGGGDEAAPPVDEESPAVDEAPADEPADDATAGDYDAESARATYEQSCIACHGGDLQGASGPALVGTGLSAAEIQDIIQNGQGSMPAQNLDDDEAANLAAWLEAQ 27

The 5 strains prepared above, KCCM11016P_Δ2284::PgapA BpOF4_13740, KCCM11016P_Δ2284::PsigB BpOF4_05495, KCCM11016P_Δ2284::PgapA BpOF4_05495, KCCM11016P: Proceeding, the results are shown in Table 6.

Individual and combination gene-enhancing strains OD, lysine production capacity, sugar consumption rate measurement Strain OD FN lysine concentration FN lysine yield opponent
Sugar 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

When the BpOF4_13740 gene was expressed with the sigB promoter and the gapA promoter, the results of an increase in the rate of sugar consumption per hour of 7.1% and 42.1% compared to the control KCCM11016P_Δ2284, respectively, were confirmed. In addition, when the similar proteins BpOF4_05495 were additionally introduced as sigB and gapA promoters, the rate of sugar consumption increased by 20.2% and 36.6%, respectively. Through these two results, it was confirmed that the sugar consumption rate (g/hr) increased according to the degree of gene strengthening according to the promoter strength. Additionally, when the genes BpOF4_13740 and BpOF4_05495 were simultaneously expressed, the highest sugar consumption rate was confirmed. Specifically, the rate of sugar consumption per hour of the KCCM11016P_Δ2284::PgapA BpOF4_13740_05495 strain was improved by 45.9% compared to the control KCCM11016P_Δ2284.

KCCM11016P_Δ2284::PgapA BpOF4_13740_05495 strain ( Corynebacterium) showing increased lysine production capacity in this example As of December 13, 2019, glutamicum CM03-0885) was deposited with the Korea Microbiological Conservation Center located in Hongje-dong, Seodaemun-gu, Seoul, Korea, and was given a deposit number of KCCM12640P.

Example 7: Analysis of lysine production capacity of BpOF4_13740_05495 enhanced strain

The genes selected in Example 6 were enhanced in Corynebacterium glutamicum KCCM10770P (Republic of Korea Patent No. 10-0924065) and KCCM11347P (Republic of Korea Patent No. 10-0073610) producing L-lysine, respectively. As an enrichment method, gene addition 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 used as an electric pulse method. produced a strain of six kinds of Solarium glutamicum KCCM10770P, and KCCM11347P respectively introduced into two kinds of strains, KCCM10770P_Δ2284, KCCM10770P_Δ2284 :: PsigB BpOF4_13740_05495, KCCM10770P_Δ2284 :: PgapA BpOF4_13740_05495, KCCM11347P_Δ2284, KCCM11347P_Δ2284 :: PsigB BpOF4_13740_05495, and KCCM11347P_Δ2284 :: PgapA BpOF4_13740_05495 Did.

The produced gene-enhanced strains were cultured in the same manner as in Example 5 to measure OD, lysine production yield, and relative sugar consumption rate per hour (based on 100% sugar consumption rate per hour of KCCM10770P_Δ2284 and KCCM11347P_Δ2284, respectively), and the results were as follows. It is shown in Table 7.

Genetically enhanced strain OD, lysine production capacity, relative consumption rate measurement Strain OD FN lysine concentration FN lysine yield Relative speed of sugar consumption 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

As shown in Table 7, the effect of reducing the fermentation time due to the rate of sugar consumption was confirmed even though the OD, FN lysine concentration and the lysine production yield level were similar in the gene-enhanced strain produced in this example.

Example 8: BpOF4 _13740_05495 was introduced CJ3P Strain production and Lysine Production capacity analysis

To confirm whether the strains belonging to other Corynebacterium glutamicums producing L-lysine have the same effect as above, three mutations in the wild strain [pyc(P458S), hom(V59A), lysC(T311I) )] to introduce L-lysine into Corynebacterium glutamicum CJ3P (Binder et al. Genome Biology 2012, 13:R40), which has the ability to produce B-OF4_13740_05495 in the same manner as in Example 7 Was produced. The produced strains were named CJ3_Δ2284, CJ3_Δ2284::PsigB BpOF4_13740_05495 and CJ3_Δ2284::PgapA BpOF4_13740_05495, respectively. The control group CJ3P strain (BpOF4_13740_05495 reinforcement is not performed) and the three production strains were cultured in the same manner as in Example 5, OD, lysine production yield and relative sugar consumption rate per hour (KCCM10770P_Δ2284 and KCCM11347P_Δ2284 hourly sugar consumption rate, respectively) 100%), and the results are shown in Table 8 below:

Genetically enhanced strain OD, lysine production capacity, relative consumption rate measurement Strain OD FN lysine concentration FN lysine yield Relative sugar 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

As shown in Table 8, despite the similar OD value, FN lysine concentration and lysine production yield level in the BpOF4_13740_05495-enhanced strain, it was confirmed that the sugar consumption rate per hour improved by 60% or more.

In the above, each description and embodiment disclosed in this specification can be applied to each other description and embodiment. All possible combinations of the various elements disclosed herein are within the scope of the inventions proposed herein. In addition, the scope of the invention of the present specification cannot be said to be limited by the specific description described below, and as long as a person having ordinary knowledge in the art can recognize or confirm a number of equivalents for a specific aspect described in the present specification , These equivalents are intended to be included in the invention proposed herein.

Korea Microbial Conservation Center KCCM12640P 20191213

<110> CJ CheilJedang Corporation <120> Microorganism for L-Lysine Production with Enhanced Cytochrome C Activity and Method of L-Lysine Production Using the Same <130> DPP20195494KR <160> 27 <170> koPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer <400> 1 taatacgact cactataggg 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> R primer <400> 2 caattaaccc tcactaaa 18 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for ATCC13032 gDNA <400> 3 gtacccgggg atcctctaga atcgcaatga tagcccattc 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R primer for ATCC13032 gDNA <400> 4 ttggtcaaac ctcccctcat atgcagaaat ccacatcaat 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for ATCC13032 gDNA <400> 5 attgatgtgg atttctgcat atgaggggag gtttgaccaa 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R primer for ATCC13032 gDNA <400> 6 gcctgcaggt cgactctaga atgcatctct ggatgatgtg 40 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for gapA promoter <400> 7 attgatgtgg atttctgcat aagcctaaaa acgaccgagc 40 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> R primer for gapA promoter <400> 8 gttgtgtctc ctctaaagat tgtag 25 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for BpOF4_13735 <400> 9 atctttagag gagacacaac atggatgaaa aaagaaaagc 40 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> R primer for BpOF4_13735 <400> 10 ttggtcaaac ctcccctcat ttaacgcccc agccaaaaaa ttcc 44 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for BpOF4_13740 <400> 11 atctttagag gagacacaac atgaaaggaa gaccactttt 40 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R primer for BpOF4_13740 <400> 12 ttggtcaaac ctcccctcat ttattctgaa atagatagta 40 <210> 13 <211> 481 <212> DNA <213> Artificial Sequence <220> <223> gapA promoter <400> 13 aagcctaaaa acgaccgagc ctattgggat taccattgaa gccagtgtga gttgcatcac 60 attggcttca aatctgagac tttaatttgt ggattcacgg gggtgtaatg tagttcataa 120 ttaaccccat tcgggggagc agatcgtagt gcgaacgatt tcaggttcgt tccctgcaaa 180 aactatttag cgcaagtgtt ggaaatgccc ccgtttgggg tcaatgtcca tttttgaatg 240 tgtctgtatg attttgcatc tgctgcgaaa tctttgtttc cccgctaaag ttgaggacag 300 gttgacacgg agttgactcg acgaattatc caatgtgagt aggtttggtg cgtgagttgg 360 aaaaattcgc catactcgcc cttgggttct gtcagctcaa gaattcttga gtgaccgatg 420 ctctgattga cctaactgct tgacacattg catttcctac aatctttaga ggagacacaa 480 c 481 <210> 14 <211> 540 <212> DNA <213> Artificial Sequence <220> <223> BpOF4_13735 nucleic acid sequence <400> 14 atggatgaaa aaagaaaagc gattattata aatgaaatta agtactggcg cgaatcaaag 60 ctgcttccct cccagtattg tgatttctta ttaacgcttt attcagaagg agaggaccta 120 gagacagccg actcaggaaa gcgcttccga aacattcgga caatctattc gtttattatt 180 gttcagcttt catttgtctt tactgctctt gtcatttatt ttactgattt ttcaaatgga 240 ttgcaaatgc ttattggttt gactttttcg attattgtgt taattatagc aaaacggact 300 agggcagatg ccttttttct taaacaattt tactatttta taggggctct gatcctcttt 360 ttactaacga ttgaatgggt tgttcactac aaaagtacta ataacctttt attatcagca 420 acaatcattt tacattgcgt tttttggctc tttgcagggc tgaaatggaa aatgcgattt 480 tttacgatat ctgctatact aggactagta gtgttaggaa ttttttggct ggggcgttaa 540 540 <210> 15 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> BpOF4_13740 nucleic acid sequence <400> 15 atgaaaggaa gaccactttt accatttgcg atcatagcaa ttgtcgggat tgttgttatg 60 atttcgcttt catttattgg gttaaaccag cgtgaagcga tgcaggcaga tgaagaagga 120 gaagaagaag taactgaaat tgaagatccg gtagcagctg gagaagaatt agtgcaaact 180 tcttgtatcg gttgtcacgg tggcgattta agcggtggtg caggtcctgc cctaacgtct 240 cttgaaggtc aatacactca agaagaaatt acagatattg ttgttaatgg gattggatca 300 atgccgtcag ttaacgataa cgaagtagaa gcagacgcaa ttgcacagta tttactatct 360 atttcagaat aa 372 <210> 16 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> BpOF4_13740 amino acid sequence <400> 16 Met Lys Gly Arg Pro Leu Leu Pro Phe Ala Ile Ile Ala Ile Val Gly 1 5 10 15 Ile Val Val Met Ile Ser Leu Ser Phe Ile Gly Leu Asn Gln Arg Glu 20 25 30 Ala Met Gln Ala Asp Glu Glu Gly Glu Glu Glu Val Thr Glu Ile Glu 35 40 45 Asp Pro Val Ala Ala Gly Glu Glu Leu Val Gln Thr Ser Cys Ile Gly 50 55 60 Cys His Gly Gly Asp Leu Ser Gly Gly Ala Gly Pro Ala Leu Thr Ser 65 70 75 80 Leu Glu Gly Gln Tyr Thr Gln Glu Glu Ile Thr Asp Ile Val Val Asn 85 90 95 Gly Ile Gly Ser Met Pro Ser Val Asn Asp Asn Glu Val Glu Ala Asp 100 105 110 Ala Ile Ala Gln Tyr Leu Leu Ser Ile Ser Glu 115 120 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for sigB promoter <400> 17 attgatgtgg atttctgcat tgcagcacct ggtgaggtgg 40 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> R primer for sigB promoter <400> 18 aactggcctc ctaaattcgc ggttc 25 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for BPOF4_13740 <400> 19 gcgaatttag gaggccagtt atgaaaggaa gaccactttt 40 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for BpOF4_05495 <400> 20 gcgaatttag gaggccagtt atgaaaaagt ttttattagc 40 <210> 21 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R primer for BpOF4_05495 <400> 21 ttggtcaaac ctcccctcat ttattgagct tcaagccatg 40 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for BpOF4_05495 <400> 22 atctttagag gagacacaac atgaaaaagt ttttattagc 40 <210> 23 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> R primer for RBS insertion <400> 23 ctgtgtttcc tcctttctcc tgttattctg aaatagatag ta 42 <210> 24 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> F primer for RBS insertion <400> 24 caggagaaag gaggaaacac agatgaaaaa gtttttatta gc 42 <210> 25 <211> 280 <212> DNA <213> Artificial Sequence <220> <223> sigB promoter <400> 25 tgcagcacct ggtgaggtgg ctgagccggt gattgaaaag attgcacaag gtttacgtga 60 gcgcggaatc accgtggaac aaggacgatt cggcgcaatg atgaaggtca catcggttaa 120 cgaaggcccc ttcaccgttt tggtcgagtg ctagccagtc aatcctaaga gcttgaaacg 180 ccccaatgtg ggggtgttaa gaactccata aaagcgcttg ggaacttttt gtggaagcag 240 tccgttgaac ctcttgaacc gcgaatttag gaggccagtt 280 <210> 26 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> BPOF4_05495 nucleic acid sequence <400> 26 atgaaaaagt ttttattagc tcttggcgca gttgttgctc ttacagcatg tggcggcgga 60 gacgaagctg ctccaccggt tgatgaggag tctccagcag tagatgaagc tccagcagat 120 gagcctgcag atgatgcaac agctggtgat tacgatgcag aatcagctcg tgctacatat 180 gagcaaagct gtatcgcatg tcatggcggc gatcttcaag gggcatcagg tccagctcta 240 gtaggaactg gcctgtcagc tgctgaaatt caagacatca tccaaaacgg acaaggttca 300 atgcctgctc aaaatttaga tgatgacgaa gctgctaacc tagctgcatg gcttgaagct 360 caataa 366 <210> 27 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> BPOF4_05495 amino acid sequence <400> 27 Met Lys Lys Phe Leu Leu Ala Leu Gly Ala Val Val Ala Leu Thr Ala 1 5 10 15 Cys Gly Gly Gly Asp Glu Ala Ala Pro Pro Val Asp Glu Glu Ser Pro 20 25 30 Ala Val Asp Glu Ala Pro Ala Asp Glu Pro Ala Asp Asp Ala Thr Ala 35 40 45 Gly Asp Tyr Asp Ala Glu Ser Ala Arg Ala Thr Tyr Glu Gln Ser Cys 50 55 60 Ile Ala Cys His Gly Gly Asp Leu Gln Gly Ala Ser Gly Pro Ala Leu 65 70 75 80 Val Gly Thr Gly Leu Ser Ala Ala Glu Ile Gln Asp Ile Ile Gln Asn 85 90 95 Gly Gln Gly Ser Met Pro Ala Gln Asn Leu Asp Asp Asp Glu Ala Ala 100 105 110 Asn Leu Ala Ala Trp Leu Glu Ala Gln 115 120

Claims (11)

A L-amino acid producing microorganism having enhanced activity of cytochrome c-551 comprising the amino acid sequence of SEQ ID NO: 16 and cytochrome c-551 comprising the amino acid sequence of SEQ ID NO: 27,
The microorganism is Corynebacterium glutamicum ,
L-amino acid producing microorganism. The method of claim 1, wherein the activity enhancement of cytochrome C encodes a cytochrome c-551 comprising the amino acid sequence of SEQ ID NO: 16 and a cytochrome c-551 comprising the amino acid sequence of SEQ ID NO: 27. L-amino acid-producing microorganisms, due to gene introduction. The microorganism of claim 1, wherein the microorganism is Corynebacterium glutamicum with an increased sugar consumption rate compared to unmodified Corynebacterium glutamicum without enhanced cytochrome C activity. delete The microorganism according to any one of claims 1 to 3, wherein the microorganism has increased L-amino acid production capacity compared to an unmodified Corynebacterium glutamicum having no enhanced cytochrome C activity. Tamicumin, a microorganism producing L-amino acids. The L-amino acid producing microorganism according to claim 5, wherein the L-amino acid is L-lysine. Cultivating the microorganism producing L-amino acid of any one of claims 1 to 3 in a medium, and
Recovering L-amino acids from the cultured microorganism, medium, or both
The production method of L-amino acid containing. The method of claim 7, wherein the L-amino acid is L-lysine. delete delete delete

Images