RU2819270C1 - Microorganism for producing l-amino acid, having high activity of cytochrome c, and method of producing l-amino acid using same microorganism - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention discloses an L-lysine producing microorganism of the genus Corynebacterium, having increased activity of cytochrome C compared to an unmodified microorganism, and a method of producing L-lysine using said microorganism. Said microorganism contains the cccA gene coding the amino acid sequence SEQ ID NO: 16 cytochrome C-551, and/or cccB gene coding amino acid sequence SEQ ID NO: 27 cytochrome C-551, where cytochrome C-551 is of origin from the microorganism Bacillus pseudofirmus OF4.

EFFECT: invention provides higher production of L-lysine.

4 cl, 1 dwg, 8 tbl, 8 ex

Description

Technical field

Cross reference to related applications

This application claims priority to KR 10-2019-0173087 and KR 10-2019-0173088, filed on December 23, 2019 with the Korea Intellectual Property Office, the entire disclosure of which is incorporated herein by reference.

An L-amino acid-producing microorganism with increased cytochrome C activity and a method for producing L-amino acid using it are proposed.

Prior Art

Microorganisms belonging to the genus Corynebacterium are Gram-positive and are widely used in the production of L-amino acids. L-amino acids, especially L-lysine, are used in the production of animal feed, in the medical and cosmetic industries. L-amino acids for industrial use are mainly produced through fermentation using Corynebacterium strains.

Many attempts have been made to improve methods for producing L-amino acids using Corynebacterium spp. Among these are studies on recombinant DNA technology, whereby specific genes are knocked down or reduced in expression to produce L-amino acids. In addition, studies have taken place in which each of the genes involved in L-amino acid biosynthesis has been amplified and analyzed for its effect on L-amino acid production by modifying L-amino acid-producing Corynebacterium strains accordingly.

In the lysine fermentation industry, obtaining high lysine concentrations and improved microbial lysine production potential are important factors. Based on this, attempts have been made to increase the lysine production potential of microorganisms and to stably maintain the improved lysine production potential during culture fermentation. However, it is difficult to maintain the lysine production potential until the late phase of cultivation due to various internal and external factors that suppress microbial activity.

Thus, there is still a need to develop a strain whose L-lysine production potential is improved and can be stably maintained.

Description of the invention

Technical problem

In one embodiment, an L-amino acid producing microorganism is provided that has increased cytochrome C activity. For example, increased cytochrome C activity can be achieved by introducing a gene encoding cytochrome C. In particular, the gene encoding cytochrome C may be an exogenous gene.

In another embodiment, a method for producing L-amino acid is provided, comprising the step of cultivating an L-amino acid-producing microorganism.

In yet another embodiment, a composition is provided for producing an L-amino acid or improving the production of an L-amino acid in a microorganism, comprising a gene encoding cytochrome C, a recombinant vector carrying this gene, or both.

Technical solution

In one embodiment, a technology for modifying a strain to produce an amino acid is provided based on a study of how amplification of a gene involved in lysine production by Corynebacterium spp. affects their lysine production potential. In general, strategies for increasing the production potential of amino acids such as lysine and the like include improving the production yields of amino acids such as lysine and the like, or increasing the production of amino acids such as lysine and the like per unit time (productivity). In particular, the amino acid productivity of lysine and so on can be affected by various factors, including components of fermentation media, osmotic pressure of fermentation media, stirring speed, oxygen supply rate and so on. Over the course of the culture period, microorganisms steadily decline in lysine production potential and cellular activity due to problems such as stress caused by various substances and metabolites present in the fermentation broth, oxygen depletion associated with increasing microbial mass, physical conditions such as temperature and mixing speed. According to one embodiment of the present invention, a technology for modifying a strain is provided to overcome the stress caused by such various factors and to enable the microorganisms to maintain constant production activity for the target products until the late phase of cultivation.

Below is a detailed disclosure of the present invention.

In one embodiment, an L-amino acid producing microorganism is provided that has enhanced cytochrome C activity.

The term "L-amino acid producing microorganism" as used herein may refer to a microorganism that has an L-amino acid producing potential that is increased as a result of an increase in its cytochrome C activity relative to its original level and/or generated from zero activity by an increase in activity of cytochrome C. The term "microorganism" as used herein may be intended to cover single-celled bacteria and may be used interchangeably with "cell".

The L-amino acid may be L-lysine.

In this document, a microorganism before its cytochrome C activity is increased may be referred to as a host microorganism to distinguish it from an “L-amino acid-producing microorganism” that has an L-amino acid producing potential that is increased or generated by increasing cytochrome C activity.

In a specific embodiment, the host microorganism can be any microorganism having the potential to produce L-amino acid (eg L-lysine). In a particular embodiment, the host microorganism may be a microorganism that has L-lysine production potential naturally or is generated by introducing a mutation into a parent strain that initially had no or very low L-lysine production potential.

In a specific embodiment, the host microorganism may be a Gram-positive bacteria that has L-lysine production potential naturally or is generated by introducing a mutation into a parent strain that initially had no or very low L-lysine production potential and, for example, may be selected from the group consisting of microorganisms of the genus Corynebacterium and microorganisms of the genus Escherichia. Examples of microorganisms of the genus Corynebacterium may include, but are not limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes and Corynebacterium efficiens. For example, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

As used herein, the term “cytochrome C” may refer to membrane-bound monomeric cytochrome C that is of bacterial origin, has an average molecular weight of 15 kDa or less, such as from about 8 kDa to about 15 kDa, and/or varies in length from 90 to 150 amino acids, from 100 to 150 amino acids, from 120 to 150 amino acids, from 90 to 125 amino acids, from 100 to 125 amino acids, or from 120 to 125 amino acids. In one embodiment, cytochrome C may be derived from microorganisms of the genus Bacillus and may be at least one selected from the cytochrome C family of proteins that exhibits the smallest energy absorption band (ability to absorb energy) at a wavelength of 550 to 555 nm or 550 to 551 nm in its reduced state. In one embodiment, cytochrome C may include at least one, such as one, two or three proteins selected from the group consisting of cytochrome C-551 (capacity to absorb energy at about 551 nm) and cytochrome C-550 (capacity to absorb energy at about 550 nm), both originating from the microorganism Bacillus (the numbers assigned at the end to cytochrome C indicate the wavelength at which cytochrome C exhibits this wavelength in its reduced state). The microorganism of the genus Bacillus may be one or more selected from the group consisting of Bacillus pseudoflrmus, Bacillus subtilis and the like.

In a specific embodiment, cytochrome C, for example at least one selected from cytochrome C-551 and cytochrome C-550, may contain an amino acid sequence encoded by CCA or CCB. In one embodiment, cytochrome C may be at least one selected from the group consisting of cytochrome C-551 derived from Bacillus pseudofirmus (e.g. Bacillus pseudofirmus OF4, etc.) and/or Bacillus subtilis, and cytochrome C-550, originating from Bacillus subtilis, and the like.

In more detail, cytochrome C (e.g., Bacillus subtilis-derived cytochrome C-551) may comprise a polypeptide comprising the amino acid sequence (e.g., SEQ ID NO: 16) encoded by CCSA (e.g., BpOF4_13740, Bacillus pseudofirmus OF4-derived), polypeptide, containing the amino acid sequence (eg SEQ ID NO: 27) encoded by CCB (eg BpOF4_05495, derived from Bacillus pseudofirmus OF4), or both.

Unless otherwise stated, cytochrome C described herein is considered to refer to any protein having sequence identity of 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 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 (for example, 60% to 99.5%, from 70% to 99.5%, from 80% to 99.5%, from 85% to 99.5%, from 90% to 99.5%, from 91% to 99.5%, from 92% to 99, 5%, from 93% to 99.5%, from 94% to 99.5%, from 95% to 99.5%, from 96% to 99.5%, from 97% to 99.5%, from 98 % to 99.5% or from 99% to 99.5%) with amino acid sequence SEQ ID NO: 16 or 27.

In particular, proteins having sequence identity falling within the scope of the cytochrome C definition described herein may be a protein having:

(1) at least one of the above characteristics of cytochrome C, for example selected from (a) bacterial origin, (b) an average molecular weight of 15 kDa or less, for example from about 8 kDa to about 15 kDa and/or a length from 90 up to 150, 100 to 150, 120 to 150, 90 to 125, 100 to 125, or 120 to 125 amino acid residues, (c) membrane binding properties, and (d) monomeric properties; and/or

(2) an effect related to an increase in activity in a microorganism having (e) an increase in L-amino acid (eg L-lysine) production potential, and/or (f) an increase in the rate of sugar consumption compared to unmodified microorganisms, where these increases are equivalent to those for proteins having amino acid sequences SEQ ID NO: 16 or 27.

In a specific embodiment, an L-amino acid producing microorganism having increased cytochrome C activity may have an increased L-amino acid production potential compared to unmodified microorganisms of the same species that have not been modified to increase cytochrome C activity.

As used herein, the term "increasing cytochrome C activity" can refer to any manipulation of a microorganism to increase its cytochrome C activity relative to the microorganism's intrinsic activity or pre-manipulation activity, including introducing cytochrome C activity into the microorganism. "Administration" may refer to the act by which cytochrome C activity is naturally or artificially generated in a microorganism that does not initially possess cytochrome C activity. The term "unmodified microorganism" may refer to a host microorganism in which cytochrome C activity is not increased (eg to a host microorganism that has not been modified to increase cytochrome C activity) or to a host microorganism that has not yet been subjected to an increase in cytochrome C activity. The term "intrinsic activity" may refer to cytochrome C activity that is maintained by the host microorganism, in in which the activity of cytochrome C is not increased, or by a host microorganism that has not yet been subjected to an increase in the activity of cytochrome C. In this context, the term "unmodified" can be used in the same sense as a condition in which a genetic modification has not been induced, but own activity is maintained.

For example, an increase in cytochrome C activity can be achieved by administering exogenous cytochrome C or by enhancing intrinsic cytochrome C activity. In a particular embodiment, an increase in cytochrome C activity can be achieved by administering exogenous cytochrome C.

In a particular embodiment, the increase in cytochrome C activity in a microorganism may be due to an increase in the rate of sugar consumption in that microorganism compared to unmodified microorganisms in which cytochrome C activity is not increased. In particular, a microorganism with increased cytochrome C activity, as contemplated in a particular embodiment, may be similar to an unmodified microorganism in terms of growth rate (OD value) and/or production yield of L-amino acid, e.g. L-lysine, over a given period of time. growth period, but shows an increased rate of sugar consumption compared to an unmodified microorganism, suggesting that a microorganism with increased cytochrome C activity produces more L-amino acid in a shorter time compared to unmodified microorganisms, thereby demonstrating improved productivity L-amino acid.

In one embodiment, increasing the activity of cytochrome C can be achieved by increasing the expression of cytochrome C at the gene (mRNA) level and/or at the protein level, and/or the activity of the cytochrome C protein itself, but not limited to them.

In one embodiment, increasing the activity of cytochrome C can be achieved by introducing a gene encoding cytochrome C. As such, introducing a gene encoding cytochrome C can increase the L-amino acid production potential that the microorganism already has, or generate an L-amino acid production potential, which the microorganism does not possess.

In one embodiment, cytochrome C or the gene encoding it may be derived from a host microorganism (homogeneous) or another microorganism (exogenous). In a specific embodiment, increasing the activity of cytochrome C can be accomplished by introducing into the host microorganism an exogenous gene encoding cytochrome C. Cytochrome C is as described above and may, for example, be cytochrome C derived from Bacillus pseudofirmus OF4 (cytochrome C-551), which is represented by the amino acid sequence SEQ ID NO: 16 (for example, encoded by CCAA (BpOF4 13740)) or the amino acid sequence SEQ ID NO: 27 (for example, encoded by CCB (BpOF4_05495)).

In one embodiment, a gene encoding cytochrome C, or an L-amino acid producing microorganism having the gene introduced therein, may comprise a polynucleotide encoding the amino acid sequence of SEQ ID NO: 16, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 27, or a combination thereof . In one embodiment, the L-amino acid producing microorganism may be a microorganism of the genus Corynebacterium, for example, Corynebacterium glutamicum, which contains a polynucleotide encoding the amino acid sequence of SEQ ID NO: 16, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 27, or a combination thereof. For example, the L-amino acid producing microorganism may be the microorganism deposited with accession number KCCM12640P.

With respect to a polynucleotide (used interchangeably with “gene”) or polypeptide (used interchangeably with “protein”), when used herein “comprising a particular nucleic acid or amino acid sequence”, “consisting of a particular nucleic acid or amino acid sequence " and "expressed as a particular nucleic acid or amino acid sequence" are interchangeable expressions, equivalently meaning that the polynucleotide or polypeptide substantially contains a particular nucleic acid or amino acid sequence. In addition, these statements may be construed as “containing a substantially equivalent sequence” (or as “not excluding the introduction of the following mutation”) that results from a mutation (deletion, substitution, modification and/or insertion) in a particular nucleic acid or amino acid sequence to the extent that the polynucleotide or polypeptide retains its original function and/or desired function.

In one embodiment, the proposed nucleic acid or amino acid sequence may include mutants obtained through conventional mutagenesis techniques, such as direct evolution and/or site-directed mutagenesis, to the extent that the mutants retain the original function or desired function of the sequence. In one embodiment, the expression that a polynucleotide or polypeptide "contains or consists of a particular nucleic acid or amino acid sequence" may mean that the polynucleotide or polypeptide essentially contains or consists of (1) a particular nucleic acid or amino acid sequence or ( 2) a nucleic acid or amino acid sequence having a sequence identity of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more (for example, from 60 % to 99.5%, from 70% to 99.5%, from 80% to 99.5%, from 85% to 99.5%, from 90% to 99.5%, from 91% to 99.5 %, from 92% to 99.5%, from 93% to 99.5%, from 94% to 99.5%, from 95% to 99.5%, from 96% to 99.5%, from 97% to 99.5%, from 98% to 99.5%, or from 99% to 99.5%), wherein the polynucleotide or polypeptide retains its original function and/or desired function. As used herein, the term "original function" means the function of cytochrome C itself (for an amino acid sequence) or the function of encoding a protein having a cytochrome C function (for a nucleic acid sequence), and the term "desired function" means the function of increasing production potential L-amino acids (eg L-lysine) in a microorganism or providing the microorganism with the potential to produce an L-amino acid (eg L-lysine).

For the nucleotide sequences described herein, various modifications may be made to the coding regions to the extent that they do not alter the amino acid sequences and/or functions of the protein (cytochrome C) expressed from the coding regions due to codon degeneracy or due to codons preferred by the microorganisms in which the protein should be expressed.

The term "identity" as used herein may refer to the degree of identity between given nucleic acid or amino acid sequences, which may be expressed as a percentage (%). The percentage identity between nucleic acid sequences can be determined using, for example, the BLAST (a program for detecting protein sequence similarity by local alignment) algorithm (see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA from Pearson (see Methods Enzymol., 183, 63 (1990)). Programs called BLASTN and BLASTX were developed based on the BLAST algorithm (see http://www.ncbi.nlm.nih.gov).

In one embodiment, a polynucleotide containing a particular nucleic acid sequence as proposed may be construed as comprising a polynucleotide containing a nucleic acid sequence complementary to that particular nucleic acid sequence, as well as a polynucleotide containing or substantially equivalent to that particular nucleic acid sequence nucleic acid sequence. More specifically, complementary polynucleotides can hybridize at suitably adjusted Tm values, for example at Tm of 55°C, 60°C, 63°C, or 65°C, in accordance with the objectives and can be analyzed under conditions that are more specifically described in known documents. For example, a condition may be mentioned in which hybridization is carried out between genes if their homology is 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, 98% or more, 98% or more, 99.5% or more, or 99.9% or more, but not carried out if their homology is below these values, or a typical condition for Southern hybridization in which one or more, more specifically two or three washes are performed at a temperature and salt concentration of 60°C, lx SSC (sodium citrate saline solution ) and 0.1% (w/v) SDS (sodium dodecyl sulfate); 60°C, 0.1x SSC and 0.1% SDS; or 68°C, but not limited to 0.1x SSC and 0.1% SDS. For hybridization, two polynucleotides must have sequences that are complementary to each other. Depending on the stringency of the hybridization, a pairing error or errors between bases may be allowed. The term "complementary" can be used to describe the relationship between nucleotide bases that can pair with each other. For DNA, for example, adenosine is complementary to thymine, and cytosine is complementary to guanine. Suitable hybridization stringency for polynucleotides may vary depending on various factors including polynucleotide length and complementarity, as is well known in the art (see Sambrook et al., 9.50-9.51, 11.7-11.8).

Increased cytochrome C activity can be achieved at the cytochrome C gene (mRNA) level through the following strategies:

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

2) modification of an expression regulatory element (sequence) in such a way as to enhance the expression of this polynucleotide, or

3) simultaneously (1) and (2), but without limitation by them.

The strategy (1) for increasing the copy number of a polynucleotide can be implemented by, but not limited to, introducing the polynucleotide into a host microorganism using a vector or introducing the polynucleotide into the chromosome of a host microorganism. As an example, increasing copy number can be accomplished by introducing into the host microorganism a polynucleotide encoding exogenous cytochrome C, or a variant polynucleotide that is codon-optimized for that polynucleotide. Any exogenous polynucleotide may be used in the present invention, without limitation as to its origin or sequence, provided that the polypeptide it encodes exhibits identical or similar activity to cytochrome C. One skilled in the art will be able to adapt and/or modify transformation methods as appropriate , known in the art for carrying out such administration. Once introduced into a host microorganism, the polynucleotide is expressed to generate exogenous cytochrome C.

The strategy (2) of modifying an expression control sequence to enhance expression of a polynucleotide can be applied to both endogenous and exogenous polynucleotides. To increase expression regulatory activity, the expression regulatory sequence may be modified by deletion, insertion, conservative or non-conservative nucleotide substitution, or a combination thereof. Alternatively, the expression control sequence may be replaced by a stronger substituent, but is not limited to this. The expression control sequence may be at least one selected from the group consisting of a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a transcription and/or translation termination control sequence. In one embodiment, instead of an endogenous promoter, a strong exogenous promoter may be operably attached upstream of the polynucleotide expression unit. Examples of a strong promoter include the CJ7 promoter, the lysCP1 promoter, the EF-Tu promoter, the groEL promoter, and the ace A or aceB promoter. More specifically, the strong promoter may be the lysCP1 promoter (WO 2009/096689) or the CJ7 promoter (WO 2006/065095), which are derived from, but are not limited to, the genus Corynebacterium.

Further described herein more specifically are instances in which a gene encoding cytochrome C is introduced into a host microorganism via a vector or inserted into the chromosome of the microorganism. Gene introduction disclosed herein can be accomplished by (1) introducing an exogenous gene (derived from a species heterologous and/or homologous to, but distinct from, the host microorganism) into a host cell using a recombinant vector carrying the gene operably linked thereto, or (2) insertion (eg, random insertion) of an exogenous gene into the chromosome (genome) of the host cell. In the case of (2) insertion into the chromosome (genome) of the host cell, such insertion can be carried out at a site that is not related to the growth of the host cell (for example, a non-transcriptional spacer (NTS), etc.), and/or the efficiency can be increased random insertion (for example, retrotransposon, etc.), but not limited to this.

To introduce a gene or vector, one skilled in the art may appropriately select a transformation method known in the art. As used herein, the term “transformation” may refer to the act of introducing a vector carrying a polynucleotide encoding a target protein (cytochrome C) into a host microorganism to express the protein encoded by the polynucleotide in the host cell. The introduced polynucleotide may be located within or outside the chromosome of the host microorganism with the possibility of expression in the host microorganism. In addition, the polynucleotide contains DNA or RNA encoding the target protein. Any delivery vehicle can be used as long as it allows for the introduction of the polynucleotide into the host microorganism and its expression. For example, the polynucleotide may take the form of an expression cassette that contains all elements necessary for autonomous expression in order to introduce the polynucleotide into a host cell. The expression cassette may typically contain expression regulatory elements operably linked to the polynucleotide, such as a promoter, a transcription stop signal, a ribosome binding site, and/or a translation stop signal. The expression cassette may be an expression vector that can replicate itself. In addition, the polynucleotide itself can be introduced into a host cell and can be operably linked to a sequence required for expression in the host cell. As used herein, the term “operably linked” means a functional link between an expression regulatory element (eg, a promoter) and a polynucleotide such that the expression regulatory element can control (eg, initiate) transcription of the polynucleotide. Making the connection in a functional manner can be accomplished using genetic recombination technology known in the art, such as, but not limited to, conventional site-specific DNA cleavage and ligation.

Any route of administration that allows the polynucleotide to transform a host microorganism can be used. Transformation technologies known in the art can be suitably selected depending on the host microorganism. Examples of transformation technologies known in the art may include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG)-mediated capture, diethylaminoethyl-dextran (DEAE)-mediated delivery, cationic liposomes, lipofection and the lithium acetate-DMSO (dimethyl sulfoxide) method, but not limited to them.

One skilled in the art can select an appropriate method for introducing a gene into a genome (chromosome) in a cell. For example, administration may be accomplished using an RNA-guided endonuclease system (e.g., at least one selected from the group consisting of a mixture of (a) RNA-guided endonuclease (e.g., Cas9 protein, etc.), a gene encoding it, or a vector carrying that gene; and (b) a guide RNA (i.e., a single guide RNA (sgRNA), etc.), the DNA encoding it, or a vector carrying that DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA), a complex (e.g., a ribonucleoprotein ( RNP) and a vector carrying a recombinant vector (for example, a gene encoding an RNA-guided endonuclease and DNA encoding a guide RNA, etc.)), but not limited to them.

According to another embodiment, the use of a gene encoding cytochrome C, a recombinant vector carrying (containing) this gene, and/or a cell carrying (containing) this recombinant vector is proposed to increase the L-amino acid production potential in a microorganism and/or imparting the L-producing potential to the microorganism -amino acids and/or in obtaining a microorganism having the potential to produce L-amino acids.

In another embodiment, a composition for producing an L-amino acid is provided, comprising a gene encoding cytochrome C, a recombinant vector carrying this gene, or a cell carrying this recombinant vector.

In another embodiment, a composition for producing an L-amino acid is provided, comprising a gene encoding cytochrome C, a recombinant vector carrying this gene, or a combination thereof. The L-amino acid producing composition can be used to provide a microorganism with the ability to produce L-amino acid, to increase the L-amino acid production potential, and/or to impart the L-amino acid production potential.

In another embodiment, a method is provided for increasing the L-amino acid production potential of a microorganism or imparting the L-amino acid production potential to a microorganism, comprising the step of introducing (transforming) a gene encoding cytochrome C, or a recombinant vector carrying this gene, into the microorganism.

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

As used herein, the term “vector” can refer to a DNA construct containing a nucleotide sequence encoding a target protein that is operably linked to a suitable regulatory sequence capable of causing expression of the target protein in a suitable host. Such regulatory sequences comprise a promoter for carrying out transcription, a possible operator sequence for regulating such transcription, a sequence encoding suitable ribosome binding sites on the mRNA, and/or sequences that regulate the termination of transcription and/or translation. Once transformed into a suitable host, the vector can replicate and function to express the target protein independently of the host genome or can be integrated into the genome itself.

Any vector that replicates in a host cell can be used without special restrictions. It can be selected from among commonly used vectors. Examples of such commonly used vectors may include plasmids, cosmids, viruses and bacteriophages, which may be in a natural or recombinant state. For example, examples of a phage vector or cosmid vector are pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A. Plasmid vectors can be derived from the pBR-, pUC-, pBluescriptII-, pGEM-, pTZ-, pCL- and pET lines. Examples of the vector may include, but are not limited to, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC.

A possible vector may be a known expression vector and/or a vector for inserting a polynucleotide into the chromosome of a host cell. Incorporation of a polynucleotide into a host cell chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. The vector may further carry a selectable marker to determine whether the gene of interest is integrated into the chromosome. The selectable marker is intended to select cells transformed by the vector, that is, to determine the incorporation of the polypeptide, and may be selected from among genes that confer selectable phenotypes such as drug resistance, auxotrophy, cytotoxic drug resistance, and surface protein expression. Under conditions where cells are exposed to a selection agent, only cells capable of expressing the selection marker can survive or exhibit a characteristic phenotype, whereby such transformed cells can be selected.

In another embodiment, a method for producing L-amino acid is provided, comprising the step of cultivating an L-amino acid-producing microorganism in a medium. The method may further include the step of isolating the L-amino acid from the cultured microorganism, the medium, or both, after the cultivation step.

In this method, the step of culturing the microorganism can be carried out by known batch culture methods, continuous culture methods, fed-batch culture methods and so on, but not particularly limited to them. Culture conditions can be maintained at optimal pH (eg pH 5 to 9, particularly pH 6 to 8, and most particularly pH 6.8) with basic compounds (eg sodium hydroxide, potassium hydroxide or ammonia) and acidic compounds (eg phosphoric acid or sulfuric acid) or under aerobic conditions by introducing oxygen or an oxygen-containing gas mixture into the cell culture, but without particular limitation thereto. The culture temperature can be maintained at 20-45°C, and specifically at 25-40°C, and the cells can be cultured for about 10 to 160 hours, but not limited thereto. The L-amino acid (ie L-lysine) produced as a result of culture may be released into the culture medium or remain within the cells.

The medium suitable for culture may contain at least one selected from sugar and carbohydrate (eg glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oil and fat (eg soybean oil, sunflower oil, peanut oil and coconut oil), fatty acid (eg palmitic acid, stearic acid and linoleic acid), alcohol (eg glycerol and ethyl alcohol) and organic acid (eg acetic acid), as a carbon source; at least one selected from nitrogen-containing organic compounds (eg peptone, yeast extract, meat juice, malt extract, corn extract, soybean meal powder and urea), inorganic compounds (eg ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and nitrate ammonium) as a source of nitrogen; at least one selected from potassium dihydrogen phosphate, potassium hydrogen phosphate or a corresponding sodium salt as a phosphorus source; and at least one selected from other essential growth promoting substances, such as, but not limited to, metal salts (eg magnesium sulfate or ferrous sulfate), amino acids and/or vitamins.

In the L-amino acid (ie L-lysine) isolation step, the target amino acid can be collected from the medium, culture or microorganisms using a suitable method known in the art, depending on the culture method. As an example, the isolation step can be carried out using at least one method selected from centrifugation, filtration, anion exchange chromatography, crystallization and HPLC (high performance liquid chromatography), and the target amino acid can be isolated from the medium or microorganism using any suitable method, known in the art. The method may further include a purification step before, simultaneously with, or after the isolation step.

Beneficial effects

Introduction of an exogenous gene provides an increase in lysine production activity in the lysine-producing strain and maintains the increased lysine production activity until a later growth phase, thereby improving the lysine production potential.

Description of graphic materials

The drawing is a schematic diagram derived from the results of analysis of nucleic acid sequences of a library of vectors obtained in one embodiment.

Carrying out the invention

The present invention will now be described in more detail by way of examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. It will be apparent to one skilled in the art that the examples described below can be modified without departing from the spirit of the invention.

EXAMPLE 1: Construction of a vector library for gene delivery

To study genes useful for increasing the lysine production potential of Corynebacterium glutamicum, a library of genomic DNAs was constructed from extremophilic bacteria that adapt to and survive various extreme environmental conditions. Four representative microorganisms, Bacillus atrophaeus (ATCC 49337), Bacillus licheniformis (KCTC 1030), Lactobacillus fermentum (KCTC 3112) were used as extremophilic bacteria that can grow under extreme conditions of high osmotic pressure, high temperatures, hypoxia and various concentrations of hydrogen ions. and Bacillus pseudofirmus OF4 (ATCC BAA2126).

First, genomic DNAs were extracted from these four strains using the QIAamp DNA Micro Kit (QIAGEN). The genomic DNAs thus obtained were digested with Sau3A1 restriction enzyme (NEB) at 37°C for 10 min and then at 65°C for 30 min to obtain crude gene fractions, which were then subjected to electrophoresis on a 1% agarose gel. Only the gel fragment in the 5 to 7 kb band was recovered. (thousand base pairs). Gene fragments for insertion were eluted from the gel using the GeneAll Expin GEL SV kit (Seoul, KOREA).

The gene fragments thus obtained were incubated with the restriction enzyme BamHI-HF (NEB) at 37°C for one hour and then with CIP (NEB) at 37°C for 30 minutes, after which they were ligated with the pECCG117 vector (Korea Patent No. 0057684). The resulting recombinant vector was transformed into E. coli DH5α, which was then applied to a plate with LB medium containing kanamycin (25 mg/l). Genes from a single colony were amplified by PCR (polymerase chain reaction) using primers SEQ ID NO: 1 and 2 shown in Table 1 below. PCR began with an initial 10-minute denaturation at 95°C and carried out 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 4 min, followed by a final extension at 72°C for 10 minutes.

Detection of PCR products confirmed that genomic DNA (gDNA) fragments obtained from an extremophilic bacterium and ranging in size from 3 to 5 kb were successfully integrated into the pECCG117 vector with a success rate of 99% or higher. Approximately 10,000 transformant colonies per strain were obtained. Plasmids were extracted from these transformants using a plasmid extraction kit (QIAGEN). The library vectors were designated p117-Lib.Bat (derived from Bacillus atrophaeus), p117-Lib.Bli (derived from Bacillus licheniformis), p117-Lib.Lfe (derived from Lactobacillus fermentum), and p117-Lib.Bps ( originates from Bacillus pseudofirmus OF4).

EXAMPLE 2: Creation and screening of a strain having a vector library introduced into it

The four library vectors (p117-Lib.Bat, p117-Lib.Bli, p117-Lib.Lfe and p117-Lib.Bps) constructed in Example 1 were used to transform the lysine-producing strain Corynebacterium glutamicum KCCM11016P (Korea Patent No. 10-0159812) by electroporation (Van der Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999), and the bacteria were applied to a complex nutrient medium containing kanamycin (25 mg/l). Ultimately, approximately 5000 colonies per library vector were obtained and designated LYS_Lib.Bat, LYS_Lib.Bli, LYS_Lib.Lfe, and LYS_Lib.Bps, respectively. Strain KCCM11016P, which was transformed with the vector pECCG117, which does not carry gDNA-derived gene fragments, was used as a control for comparison with library strains and was designated control LYS_117.

The composition of the complex nutrient medium used was as follows.

Complex nutrient medium (pH 7.0):

glucose 10 g, peptone 10 g, meat extract 5 g, yeast extract 5 g, brain heart extract 18.5 g, NaCl 2.5 g, urea 2 g, sorbitol 91 g, agar 20 g (per liter of distilled water) .

Each of the four resulting KCCM11016P library strains was inoculated into a 96-Deep Well Plate Dome (Bioneer) containing 400 μl of screening medium using a colony picker (SINGER, PIXL) and incubated in a shaking the plates (TAITEC) at 32°C for 15 hours at a speed of 12,000 rpm.

The sowing medium has the following composition.

Screening medium (pH 7.0):

glucose 45 g, molasses obtained from sugar beets 10 g, soybean soaking liquid 10 g, (NH ₄ ) ₂ SO ₄ 24 g, MgSO ₄ ⋅ 7H ₂ O 0.6 g, KH ₂ PO ₄ 0.55 g, urea 5.5 g, biotin 0.9 mg, thiamine HCl 4.5 mg, calcium pantothenate 4.5 mg, nicotinamide 30 mg, MnSO ₄ ⋅5H ₂ O 9 mg, ZnSO ₄ ⋅5H ₂ O 0.45 mg, CuSO ₄ ⋅5H ₂ O 0.45 mg, FeSO ₄ ⋅5H ₂ O 9 mg and kanamycin 25 mg (per liter of distilled water).

During cultivation, cell growth was monitored using a microplate reader (BioTek). The concentrations of glucose and lysine production in the media were measured using a sugar analyzer (YSI) and an HPLC (high performance liquid chromatography, Shimadzu) apparatus, respectively.

During the experiment, three strains were finally selected that show excellent growth and high rates of glucose consumption compared to the control (Table 2). It was found that these three selected strains carry the library vectors LYS_Lib.Bps. Although they were similar to the LYS_117 control in terms of yield and OD values, they were found to have excellent productivity as their glucose consumption rates per hour (g/h) at the sampling point were increased to 118% compared to 100% for the LYS_117 control .

EXAMPLE 3: Base sequencing of a gDNA library

To identify the gene sequences introduced into the three colonies selected in Example 2, LYS_Lib.Bps #257, #881 and #4213, the gDNA library gene fragments contained in the colonies were amplified by PCR using primers SEQ ID NO: 1 and 2 shown in Table 1 of Example 1. PCR was performed under the same conditions as in Example 1. PCR fragments were isolated using the GeneAll Expin GEL SV kit (Seoul, KOREA) and analyzed for base sequence. Based on the analysis results, gene information was obtained via BLAST (NCBI (National Center for Biotechnology Information) reference sequence NC_013791.2).

The results of the analysis are presented in the drawing. As shown in the drawing, the result of base sequencing indicates that colony LYS_Lib.Bps #257 has a gene fragment of 4794 bp, colony #881 has a gene fragment of 3985 bp, and colony #4213 has a gene fragment of 4483 By. These three colonies were found to contain BpOF4_13735 and BpOF4_13740 as ORF (open reading frame) intact genes similar to each other. Subsequently, additional experiments were performed regarding the influence of these two genes.

EXAMPLE 4: Construction of a vector and a strain containing a separate gene introduced into it

A genomic insertion vector was constructed for use in studying the effects of the two individual genes identified in Example 3. The pDZ_Δ2284 vector targeting Ncgl2284, which is one of the transposases, was constructed to serve as the main vector for gene insertion.

More specifically, ATCC13032 gDNA was used as template DNA for PCR. Primers were prepared based on the NCBI base sequence (NC_003450.3). In the presence of primers SEQ ID NO: 3 and 4, PCR was started with a 10-minute denaturation at 95°C and 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72° were performed. C for 4 minutes followed by a final extension at 72°C for 10 minutes to obtain a 5' DNA fragment having a length of approximately 900 bp. PCR was carried out similarly using primers SEQ ID NO: 5 and 6 under the same conditions as described above to amplify the 3' DNA fragment. These two DNA amplicons were purified using the GeneAll Expin GEL SV kit (Seoul, KOREA) and digested with the restriction enzyme XbaI (NEB). Using an In-Fusion cloning kit, the digestion product was ligated to pDZ (Korea Patent No. 2009-0094433), which was heat-treated at 65°C for 20 min. The recombinant plasmid thus formed was transformed into E. coli DH5α, which was then applied to a plate of LB medium containing kanamycin (25 mg/l). The gene inserted into the pDZ vector was subjected to base sequencing to ultimately yield the pDZ_Δ2284 vector.

An additional enrichment (expression) vector for individual genes was constructed by digesting the main vector pDZ_Δ2284 with the restriction enzymes NdeI and CIP (NEB), heat treating the digested vector at 65°C for 20 min, purifying the heat-treated vector and ligating the promoter and each gene DNA fragment into vector using In-Fusion cloning kit. The gapA gene promoter with SEQ ID NO: 13 was used as a promoter for additional gene expression. To obtain the promoter, PCR was performed using primers SEQ ID NO: 7 and 8 with ATCC13032 gDNA (NC_003450.3) serving as a template. In the presence of pfu polymerase, PCR was started with 10 min of denaturation at 95°C and carried out 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, followed by final extension at 72°C for 10 min. DNA fragments for the two genes BpOF4_13735 and BPOF4_13740 were amplified from Bacillus pseudofirmus OF4 gDNA in the same manner as for the promoter, except using primers SEQ ID NO: 9 and 10 for BpOF4_13735 (SEQ ID NO: 14) and primers SEQ ID NO: 11 and 12 for BpOF4_13740 (SEQ ID NO: 15). The DNA fragments thus obtained were purified using the GeneAll Expin GEL SV kit (Seoul, KOREA) and then ligated with pDZ_Δ2284 to ultimately obtain two different vectors: pDZ_Δ2284::PgapA BpOF4_13735 and pDZ_Δ2284::PgapA BpOF4_13740.

Each of the two vector constructs was transformed into a lysine-producing strain of Corynebacterium glutamicum KCCM11016P (Korea Patent No. 10-0159812) using the electroporation method. Secondary DNA crossover enriched individual genes in the strains. The two final strains thus constructed were designated KCCM11016P_Δ2284::Pgap A BpOF4_13735 and KCCM11016P_Δ2284::PgapA BpOF4_13740, respectively.

Primers, promoters, nucleic acids used

the sequences of the BpOF4 genes and the amino acid sequences encoded by these genes are shown in Table 3 below.

EXAMPLE 5: Analysis of the lysine production potential of strains with individual genes introduced into them

The two strains obtained in Example 4 were cultured as follows to measure OD values, lysine production yields and sugar consumption rates (g/h). First, each strain was inoculated into a 250-ml corner flask containing 25 ml of seed medium and then cultured at 30°C for 20 hours with shaking at 150 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner flask containing 24 ml of production medium and then cultured at 32°C for 40 hours with shaking at 150 rpm. The compositions of the inoculation medium and production medium are given below, and the culture results are presented in Table 4 below. Sowing medium (pH 7.0):

glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ ⋅ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 mcg, calcium pantothenate 2000 mcg, nicotinamide 2000 mcg (per liter of distilled water).

Production medium (pH 7.0):

glucose 45 g, molasses obtained from sugar beet 10 g, soybean soaking liquid 10 g, (NH ₄ ) ₂ SO ₄ 24 g, MgSO ₄ ⋅ 7H ₂ O 0.6 g, KH ₂ PO ₄ 0.55 g, urea 5.5 g, CaCO ₃ 30 g, biotin 0.9 mg, thiamine HCl 4.5 mg, calcium pantothenate 4.5 mg, nicotinamide 30 mg, MnSO ₄ ⋅5H ₂ O 9 mg, ZnSO ₄ ⋅5H ₂ O 0 .45 mg, CuSO ₄ ⋅5H ₂ O 0.45 mg, FeSO ₄ ⋅5H ₂ O 9 mg and kanamycin 25 mg (per liter of distilled water).

The strain overexpressing BpOF4_13735, one of two genes obtained from the gDNA library, did not show significantly improved effects in lysine production yield and sugar consumption rate compared with the parental strain KCCM11016P_Δ2284. In contrast, strain KCCM11016P_Δ2284::PgapA BpOF4_13740, although similar to the parent strain KCCM11016P_Δ2284 in final OD value and yield, was found to have an increased rate of sugar consumption per hour during the middle culture period (17 to 24 hours). by 31.5% compared to the parent strain.

Finally, the colony effects of LYS_Lib.Bps #257, #881 and #4213 shown in Example 2 were found to be due to the upregulation of BpOF4_13740.

EXAMPLE 6: Increased Lysine Production Potential in BpOP4_13740-Enhanced Strain

In order to secondarily verify the effect of the BpOF4_13740 gene confirmed in Example 5, the BpOF4_13740 gene was analyzed after its amplification with a different promoter. And the effect for the gene BpOF4_05495, also confirmed by NCBI BLAST analysis as the same functional protein, was also analyzed.

For this purpose, a gene expression vector was further constructed in the same manner as in the case of the vector constructed in Example 3.

The sigB promoter was amplified by PCR using primers SEQ ID NOS: 17 and 18 with ATCC13032 gDNA as template. Using primers SEQ ID NOS: 19 and 12, PCR was performed on the Bacillus pseudofirmus OF4 gDNA template with amplification of the BpOF4_13740 gene. These two gene fragments were ligated into pDZ_Δ2284 to produce the vector pDZ_Δ2284::PsigB BpOF4_13740.

Amplification of the BpOF4_05495 gene was also achieved in a similar manner by constructing the vectors pDZ_Δ2284::PsigB BpOF4_05495 and pDZ_Δ2284::PgapA BpOF4_05495. The BpOF4_05495 gene fragment was obtained using primers SEQ ID NOS: 20 and 21 and primers SEQ ID NOS: 22 and 21. The effect obtained by simultaneously enriching these two genes was also evaluated. For this purpose, primers SEQ ID NOS: 23 and 24 were additionally synthesized, which were designed so that they contained the ribosome binding sequence (RBS), and the vectors pDZ_Δ2284::PsigB BpOF4_13740_05495 and pDZ_Δ2284::PgapA BpOF4_13740_05495 were constructed.

Each of five additional vector constructs (vector pDZ_Δ2284::PsigB BpOF4_13740, vector pDZ_Δ2284::PsigB BpOF4_05495, vector pDZ_Δ2284::PgapA BpOF4_05495, vector pDZ_Δ2284::PsigB BpOF4_1 3740_05495 and vector pDZ_Δ2284::PgapA BpOF4_13740_05495) transformed the lysine-producing strain Corynebacterium glutamicum KCCM11016P (Korea Patent No. 10-0159812) by electroporation and subjected to secondary DNA crossover to obtain strains containing enhanced individual genes.

These five strains thus obtained were designated as KCCM11016P_Δ2284::PsigB BpOF4_13740, KCCM11016P_Δ2284::PsigB BpOF4_05495, KCCM11016P_Δ2284::PgapA BpOF4_05495, KCCM11016P_ Δ2284::PsigB BpOF4_13740_05495 and KCCM11016P_Δ2284::PgapA BpOF4_13740_05495, respectively.

Primers, promoters, nucleic acid sequences of BpOF4_05495 and the amino acid sequence encoded by this gene are shown in Table 5 below.

These five strains, KCCM11016P_Δ2284::PsigB BpOF4_13740, KCCM11016P_Δ2284::PsigB BpOF4_05495, KCCM11016P_Δ2284::PgapA BpOF4_05495, KCCM11016P_Δ2284 ::PsigB BpOF4_13740 05495 and KCCM11016P_Δ2284::PgapA BpOF4_13740 05495 were evaluated in flasks under the same conditions as in Example 5, and the results are presented in Table 6 below.

Expression of the BpOF4_13740 gene under the control of the sigB promoter and the gapA promoter resulted in increased sugar consumption rates per hour by 7.1% and 42.1%, respectively, compared to the KCCM11016P_Δ2284 control. In addition, when the BpOF4_05495 gene, which encodes a similar protein, was additionally introduced under the control of the sigB and gap A promoters, sugar consumption rates increased by 20.2% and 36.6%, respectively. These two results suggest that sugar consumption rates (g/h) increase when the gene is amplified under promoter control. In addition, it was observed that sugar consumption rates reached maximum values when the BpOF4_13740 and BpOF4_05495 genes were simultaneously expressed. More specifically, strain KCCM11016P_Δ2284::PgapA BpOF4_13740 05495 exhibited a sugar consumption rate per hour increased by 45.9% compared to the control KCCM11016P_Δ2284.

Strain KCCM11016P_Δ2284::PgapA BpOF4_13740_05495 (designated as Corynebacterium glutamicum CM03-885), which has increased lysine production potential, was deposited in the Korean Microbial Culture Center located in Hongje-dong, Seodamun-Gu, Seoul, Korea, on December 13, 2019 with assigned we eat him access numbers KCCM 12640Р.

EXAMPLE 7: Analysis of BPOE4_13740_05495-enhanced strain for lysine production potential

Each of the L-lysine-producing Corynebacterium glutamicum strains KCCM10770P (Korea Patent No. 10-0924065) and KCCM11347P (Korea Patent No. 10-0073610) were enhanced with the genes selected in Example 6. For this purpose, the genes were introduced in the same manner as in Example 6. Ultimately, each of the three vectors pDZ_A2284, _P DZ_A2284::PsigB BpOF4_13740_05495 and pDZ_Δ2284::PgapA BpOF4_13740 05495 transformed two Corynebacterium glutamicum strains KCCM10770P and KCCM11347P to obtain a total of six 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.

The gene-enhanced strains thus obtained were cultured in the same manner as in Example 5, and the OD, lysine production yield and relative sugar consumption rate per hour (when the sugar consumption rates per hour for KCCM10770P_Δ2284 and KCCM11347P_Δ2284 were taken as 100%) were measured and the results are presented in Table 7 below.

As shown in Table 7, the gene-enhanced strains produced in this Example, although similar to the control in terms of OD, final (FN) lysine concentration and lysine production yield, had shorter fermentation times due to their higher rate of sugar consumption.

EXAMPLE 8: Preparation of strain CJ3P with introduced BpOF4_13740_05495 and its analysis regarding its lysine production potential

A study was conducted to determine whether different L-lysine producing variants of Corynebacterium glutamicum would show the same effect as shown above. For this purpose, the Corynebacterium glutamicum strain CJ3P (Binder et al. Genome Biology 2012, 13:R40), which was obtained by introducing three mutations [pyc(P458S), hom(V59A) and lysC(T311I)] into the wild type to give it the potential L-lysine production was enhanced by BpOF4_13740_05495 in the same manner as in Example 7. The enhanced strains thus obtained were designated CJ3_Δ2284, CJ3_Δ2284::PsigB BpOF4_13740_05495 and CJ3_Δ2284::PgapA BpOF4_137 40_05495, respectively. The control strain CJ3P (not enhanced with BpOF4_13740_05495) and the three resulting strains were cultured in the same manner as in Example 5, and their OD, lysine production yield, and relative sugar consumption rate per hour (where the sugar consumption rate per hour for each KCCM10770P_Δ2284 and KCCM11347P_Δ2284 was taken as 100%), and the results are presented in Table 8 below.

As can be seen from Table 8, the BpOF4_13740_05495-enhanced strain, although similar to the control in terms of OD, final (FN) lysine concentration and lysine production yield, was found to exhibit an increase in the rate of sugar consumption per hour by 60% or more.

From the above description, one skilled in the art will appreciate that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Thus, it should be understood that the embodiments described above are illustrative in all aspects and not limiting. The scope of this application is to be interpreted as being within the scope of this application, all changes or modifications derived from the meaning and scope of the appended claims and their equivalents rather than from the detailed description.

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<110> CJ CheilJedang Corporation

<120> Microorganism for L-Amino Acid Production with Enhanced

Cytochrome C Activity and Method of L-Amino Acid Production Using the

Same

<130>OPP20203644KR

<150> KR 10-2019-0173087

<151> 2019-12-23

<150> KR 10-2019-0173088

<151> 2019-12-23

<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> nucleic acid sequence BpOF4_13735

<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> nucleic acid sequence BpOF4_13740

<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

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<223> amino acid sequence of BpOF4_13740

<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

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attgatgtgg atttctgcat tgcagcacct ggtgaggtgg

40

<210> 18

<211> 25

<212> DNA

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<400> 18

aactggcctc ctaaattcgc ggttc

25

<210> 19

<211> 40

<212> DNA

<213> Artificial Sequence

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<223> F primer for BPOF4_13740

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gcgaatttag gaggccagtt atgaaaggaa gaccactttt

40

<210> 20

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> F primer for BpOF4_05495

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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> primer F 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> nucleic acid sequence BPOF4_05495

<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> amino acid sequence of BPOF4_05495

<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 (6)

1. An L-lysine-producing microorganism of the genus Corynebacterium , which has increased cytochrome C activity compared to an unmodified microorganism, containing the cccA gene encoding the amino acid sequence SEQ ID NO: 16 of cytochrome C-551, and/or the cccB gene encoding the amino acid sequence SEQ ID NO : 27 cytochrome C-551, where cytochrome C-551 is derived from the microorganism Bacillus pseudofirmus OF4. 2. An L-lysine-producing microorganism according to claim 1, which has an increased rate of sugar consumption compared to a homogeneous microorganism in which the activity of cytochrome C is not increased. 3. L-lysine-producing microorganism according to any one of claims. 1 or 2, having an improved L-lysine production potential compared to a homogeneous microorganism in which cytochrome C activity is not increased. 4. A method for producing L-lysine, including the stages: cultivating the L-lysine-producing microorganism according to claim 1 or 2 in a medium; And isolating L-lysine from the cultured microorganism, the medium, or both.