Technical Field
The present invention relates to a method for producing
high concentrations of L-amino acid and riboflavin, and a
microorganism for simultaneously producing L-amino acid and
10 riboflavin.
Background Art
Cereal grains, such as corn, Indian millet, barley and
wheat, which are most frequently used in feed, usually provide
15 30-60% of the amino acid requirement. Thus, in order to
satisfy the remaining requirements and maintain the balance
between essential amino acids, additional provision of amino
acids is required. Also, all feeds contain certain amounts of
vitamins, and if any vitamin is not provided in a sufficient
20 amount, a deficiency of the vitamin can be occurred. Thus,
vitamins should additionally be provided to maintain their
proper levels, like amino acids. Amino acids are the most
expensive among feed components, and efficient provision of
amino acids can be considered as one of factors that
25 determines the overall ability to produce livestock.
2
Particularly, L-lysine and L-threonine among amino acids
frequently become the first amino acids that limit the growth
of livestock. L-lysine and L-threonine may be produced by a
fermentation process using microorganisms, and the L-lysine
5 and L-threonine produced by the fermentation process are added
to feed after purification and concentration. Microorganisms
that are typically used in the fermentation of L-lysine are
Corynebacterium sp. or Escherichia coli, and many examples that
produced L-lysine by genetically engineering the microorganisms
10 have been reported (Korean Patent No. 10-0930203 and No. 10-
0924065, and US Patent No. 7,871,801).
Currently, efforts are being continuously made to increase
the production of L-lysine or L-threonine by modified
microorganisms using genetic engineering methods. However,
15 with the growth of the industry, the ability to provide
increased amounts of L-lysine or L-threonine is required, and
thus efforts are being made to develop methods capable of
producing L-lysine or L-threonine in a more effective and
economical way.
20 Although vitamins are required in small amounts, they are
essential organic compounds that must be provided for the
maintenance of normal metabolic functions, growth, reproductive
functions and health of livestock. Among vitamins, riboflavin
(vitamin B2) is a water-soluble vitamin that is biosynthesized
25 in various species of microorganisms and all kinds of plants,
3
but it is not biosynthesized in the body of vertebrates,
including humans, and thus is required to be provided by
external sources. A deficiency of riboflavin can cause
anestrus and reproductive failure in pigs (Biol. Reprod. (1981)
5 25:659-665, J. Anim. Sci. (1984) 59:1567-1572). In fowls, it
can cause problems in nerves, particularly sciatic nerves and
brachial nerves and can adversely affect the growth of embryos,
resulting in the death of embryos (the Korean Feeding Standard
for Poultry, 2002, the Korean Ministry of Agriculture and
10 Forestry). Thus, riboflavin has been used as a feed additive
for the growth of livestock, and particularly, concentrated
riboflavin itself has been used as feed.
The current worldwide production of riboflavin is 6,000
tons per year, of which 75% is used as feed additives and the
15 remainder is used as foods and pharmaceuticals. In riboflavin
production, a chemical synthesis method and a microbial
fermentation method are used. In a chemical synthesis method,
high-purity riboflavin is produced from a precursor such as Dribose
by a multi-step process. The chemical synthesis method
20 has a disadvantage in that the starting material is expensive
and thereby increases the production cost. For this reason, a
method of producing riboflavin by microbial fermentation was
developed. The microbial fermentation method is a method in
which either a microorganism that produces riboflavin is
25 isolated from nature or a microorganism mutated by a genetic
4
engineering method or a chemical/physical method so as to
overproduce riboflavin is cultured under suitable conditions,
and then riboflavin is isolated from the culture. In recent
years, the fermentation method has been primarily researched,
5 because it is price-competitive and environmentally friendly.
Riboflavin produced by the fermentation method is added to feed
after purification and concentration.
A typical method of producing riboflavin using the yeast
Candida famata is disclosed in US Patent No. 5,231,007. In the
10 industrial production of riboflavin, Eremothecium ashbyii and
Ashbya gossypii (WO No. 95/26406), which are belong to
Ascomycetes, are most frequently used. In addition, the
bacterium Bacillus subtilis was also reported as a strain that
can be used for producing riboflavin. Many examples that
15 produced riboflavin by genetically engineering the above
bacterium have been reported (EP No. 0821063, US Patent No.
5,837,528, and US Patent No. 5,334,510), and the present
inventors also produced riboflavin using the above bacterium
(Korean Patent No. 10-0542573). In addition, an example that
20 produced 4.5 g/L of riboflavin using a microorganism engineered
to overexpress a riboflavin biosynthesis-related enzyme gene
was also reported (J. Ind. Microbiol. Biotechnol. (1999), 22:8-
8).
The requirements for components in animal feed are about
25 1-5 g/kg of L-lysine, about 0.6-3.3 g/kg of L-threonine, and 2-
5
4 mg/kg of riboflavin, which is about 0.1% of the requirement
of L-lysine (NRC. 1998. National Academy of Sciences- National
Research Council, Washington, D.C.). However, L-lysine and
riboflavin are separately produced by fermentation processes,
5 are subjected to purification and concentration processes
before their addition to feed, and are individually transferred
to a feed compounding plant. For this reason, they can
increase the production cost of feed. If the concentration of
riboflavin in a microorganism that produces both L-lysine and
10 riboflavin reaches about 0.1% of the concentration of L-lysine,
addition of the microbial culture can satisfy the requirements
for feed additives, but attempts to achieve this have not yet
been reported.
In the case of Corynebacterium sp. microorganisms,
15 riboflavin is biosynthesized through two pathways from ribulose
5-phosphate (Ru5P), which is a pentose-phosphate pathway (PPP)
product, and guanosine triphosphate (GTP) that is a purine
metabolism product. In the biosynthesis of riboflavin, a gene
family consisting of GTP cyclohydrolase II (RibA) gene,
20 pyrimidine deaminase-reductase (RibG) gene, riboflavin synthase
subunit alpha (RibC) gene and riboflavin synthase subunit beta
(RibH) gene (hereinafter referred to as “riboflavin
biosynthesis gene family”) is involved. The riboflavin
biosynthesis gene family forms an operon (rib operon) with
25 ribulose-5-phosphate-3-epimerase (Rpe, NCgl1536) that is
6
involved in the pentose-phosphate pathway, and both the Rpe and
the RibA compete in the use of Ru5P as a substrate (FIG. 1).
In other words, Rpe biosynthesizes D-xylose-5-phosphate from
Ru5P to mediate an intermediate process in which a metabolic
5 product produced in the pentose-phosphate pathway enters the
glycolytic pathway, and RibA biosynthesizes 3,4-dihydroxy-2-
butanone-4-phosphate that is an intermediate of riboflavin
biosynthesis (KEGG, Kyoto Encyclopedia of Genes and Genomes,
[//www.genome.jp/kegg]).
10 The pentose-phosphate pathway in Corynebacterium is the
major source of reducing power (NADPH) that is involved in
lysine biosynthesis, and the direct correlation between the
regeneration of NADPH and L-lysine biosynthesis has been
reported in the literature (Wittmann and Heinzle, Microbiol
15 68:5843-5849, 2002; Marx et al., J Biotechnol 104:185-197,
2003; Ohnishi et al., Microbiol Lett 242:265-274, 2005).
The pentose-phosphate pathway is catalyzed by glucose-6-
phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase, 6-
phosphogluconate dehydrogenase (6PGD), Rpe, ribose-5-phosphate
20 isomerase (RpiA), transketolase and transaldolase, and the
final metabolic product produced by this pathway enters the
glycolytic process.
As a result of studies on the enforcement of the pentosephosphate
pathway, EP 01941065 (June, 2001) and EP 02781875
25 (December, 2002) disclose an enzyme variant which directly
7
produce NADPH, which has negative feedback resistance. In
addition, inventions relating to lysine-producing
Corynebacterium strains that overexpress transketolase and
transaldolase were disclosed (EP 1109915, EP 1179076, and EP
5 1179084). Moreover, an increase in the production of L-lysine
in a Corynebacterium strain that overexpresses Rpe or RpiA was
reported (DE10037611 and DE10037612).
Thus, it can be seen that as the production of L-lysine
in a Corynebacterium strain increases, the dependence of the
10 strain on the pentose-phosphate pathway increases. However,
the riboflavin biosynthesis pathway that is the key element of
the present invention is derived from the pentose-phosphate
pathway, and thus if a carbon flow to the riboflavin
biosynthesis pathway is enhanced, a carbon source to be
15 introduced into the glycolytic pathway through the pentosephosphate
pathway will leak, resulting in a decrease in the Llysine
production yield per unit of carbon source supplied. In
other words, it can be considered that the riboflavin
biosynthesis pathway in an L-lysine-producing strain that
20 requires a sufficient carbon flow in the pentose-phosphate
pathway is competitive with the pentose-phosphate pathway.
Also, if a carbon flow that is introduced into the riboflavin
biosynthesis pathway increases, the production of L-lysine can
be adversely affected.
25 Thus, microorganisms that simultaneously produce L-lysine
8
and riboflavin can be present in nature, but the production
yields of L-lysine and riboflavin can be competitive with each
other. For this reason, attempts to increase the production of
riboflavin in an industrial microorganism, which produces a
5 large amount of L-lysine, to an industrially useful level, have
not yet been reported.
Disclosure
Technical Problem
10 Under above circumstances, the present inventors have
made extensive efforts to develop a microorganism that
simultaneously produces L-lysine and riboflavin. As a result,
the present inventors have imparted high-concentration gluconic
acid adaptability into a Corynebacterium strain having L15
lysine-producing ability by artificial mutation in order to
increase carbon flow in the pentose-phosphate pathway, thereby
developing a mutant strain that has an increased ability to
produce riboflavin compared to the parent strain while
maintaining the production of L-lysine at a similar level to
20 that of the parent strain. Also, the present inventors have
found that, even when the promoter of the riboflavin
biosynthesis gene family in an L-lysine-producing
Corynebacterium strain is replaced with a heterogeneous strong
promoter, the Corynebacterium strain can simultaneously produce
25 L-lysine and riboflavin at high concentrations, and the same
9
effect also appears in a Corynebacterium strain having Lthreonine-
producing ability, thereby completing the present
invention.
5 Technical Solution
It is an object of the present invention to provide a
method for producing L-lysine or L-threonine, and riboflavin,
comprising: culturing a microorganism which is obtained by
modifying a Corynebacterium sp. microorganism having L-lysine
10 or L-threonine-producing ability so as to increase the
production of riboflavin while maintaining the production of Llysine
or L-threonine at a high level; and producing L-lysine
or L-threonine, and riboflavin by a fermentation process.
Another object of the present invention is to provide a
15 modified Corynebacterium glutamicum microorganism which is
obtained by inducing a random mutation in a Corynebacterium
glutamicum microorganism having high-concentration L-lysineproducing
ability so as to increase the production of
riboflavin while maintaining the production of L-lysine at a
20 high level, wherein the modified Corynebacterium glutamicum
microorganism is deposited under accession No. KCCM11223P.
Still another object of the present invention is to
provide a modified Corynebacterium sp. microorganism for
simultaneously producing L-lysine or L-threonine, and
25 riboflavin, which is modified by enhancing activity of an
10
enzyme family that is expressed by the rib operon comprising
riboflavin biosynthesis gene family in a Corynebacterium sp.
microorganism having L-lysine or L-threonine-producing ability
so as to increase the production of riboflavin while
5 maintaining the production of L-lysine or L-threonine at a high
level.
Still another object of the present invention is to
provide a formulation or granular formulation comprising Lamino
acid and riboflavin, which is prepared by culturing the
10 above-described microorganism and granulating L-amino acid and
riboflavin.
Still another object of the present invention is to
provide a feed additive comprising a formulation comprising Lamino
acid and riboflavin, which is prepared by culturing the
15 above-described microorganism, or the above-described granular
formulation.
Advantageous Effects
The present invention can provide a developed
20 microorganism that can simultaneously produce high
concentrations of L-amino acid, such as L-lysine or Lthreonine
which is industrially produced in large amounts, and
riboflavin, which are used as essential animal feed, and the
method for producing L-amino acid and riboflavin. Thus, the
25 present invention can provide the effects of manufacturing
11
feeds conveniently and reducing the production cost and can
provide an efficient strain for producing L-amino acid and
vitamin feed additives.
5 Description of Drawings
FIG. 1 shows that the glycolytic pathway and the
riboflavin biosynthesis pathway compete in the use of ribulose-
5-phosphate.
FIG. 2 shows that a strong promoter is inserted to the
10 upstream or inside of the rib operon to enhance the activity of
either the enzyme family that is encoded by the rib operon or
the riboflavin biosynthesis enzyme family.
FIG. 3 shows a pDZ-Pmrb vector constructed in order to
insert a promoter of SEQ ID NO: 5.
15 FIG. 4 shows a pDZ-lysCP1-ribG vector constructed in order
to insert a promoter of SEQ ID NO: 6.
Best Mode
Hereinafter, the terms used herein will be defined.
20 As used herein, the term "rib operon" refers to an operon
including a riboflavin biosynthesis gene family consisting of
ribG gene encoding pyrimidine deaminase-reductase (RibG,
NCgl1535), ribC gene encoding riboflavin synthase subunit alpha
(RibC, NCgl1534), ribA gene encoding GTP cyclohydrolase II
25 (RibA), and ribH gene encoding riboflavin synthase subunit beta
12
(RibH, NCgl1532) (hereinafter referred to as “riboflavin
biosynthesis gene family”), and rpe gene encoding ribulose-5-
phosphate-3-epimerase (Rpe, NCgl1536) that is involved in the
pentose-phosphate pathway (FIG. 2). Information on the genes
5 of the operon is available from public databases (e.g., NCBI
GenBank).
As used herein, the term "L-amino acid" refers to an amino
acid selected from the group consisting of L-lysine, Lthreonine,
L-valine, L-isoleucine, L-tryptophan and L10
methionine. Specifically, the amino acid is L-lysine or Lthreonine.
One aspect of the present invention contains a method for
producing L-lysine or L-threonine, and riboflavin, comprising:
15 culturing a microorganism which is obtained by modifying a
Corynebacterium sp. microorganism having L-lysine or Lthreonine-
producing ability so as to increase the production of
riboflavin while maintaining the production of L-lysine or Lthreonine
at a high level; and producing L-lysine or L20
threonine, and riboflavin by a fermentation process.
In a specific example of the present invention, it was
found that the production of L-lysine or L-threonine in the
modified microorganism was maintained at the same level as that
25 of the parent strain while the production of riboflavin
13
increased by about 70-80 times, and particularly the ratio
between the production of lysine and the production of
riboflavin was about 1:0.005 or the ratio between the
production of L-threonine and the production of riboflavin was
5 about 1:0.03 (Tables 1 to 6). This suggests that L-amino acid
and riboflavin simultaneously produced by the microorganism of
the present invention are suitable for use as feed additives.
The modified microorganism is modified Corynebacterium sp.
microorganism for simultaneously producing L-amino acid and
10 riboflavin, which is obtained by modifying a Corynebacterium
sp. microorganism having L-amino acid-producing ability so as
to increase the production of riboflavin while maintaining the
production of L-amino acid, as a result of enhancing the
activity of an enzyme family that is expressed by the rib
15 operon including the riboflavin biosynthesis gene family.
Specifically, it may be a mutant Corynebacterium sp.
microorganism for simultaneously producing L-lysine or Lthreonine
and riboflavin, which is obtained by modifying a
Corynebacterium sp. microorganism having L-lysine or L20
threonine-producing ability so as to increase the production of
riboflavin while maintaining the production of L-lysine or Lthreonine,
as a result of enhancing the activity of an enzyme
family that is expressed by the rib operon including the
riboflavin biosynthesis gene family.
25 The Corynebacterium sp. microorganism used in the present
14
invention may be any Corynebacterium sp. strain having the
ability of producing L-amino acid, and examples thereof include,
but are not limited to, Corynebacterium glutamicum (ATCC 13032),
Corynebacterium ammoniagenes, Corynebacterium thermoaminogenes
5 (FERM BP-1539), Brevibacterium flavum (ATCC 14067), and
Brevibacterium fermentum (ATCC 13869). More specifically,
Corynebacterium glutamicum may be used, and specific examples
thereof include, but are not limited to, KFCC10881 (Korean
Patent No. 0159812), KFCC11074 (Korean Patent No. 0292299),
10 KFCC11001 (Korean Patent No. 0253424) and KCCM11222P. In a
specific example of the present invention, a mutant
microorganism was produced by modifying KFCC10881 as a parent
strain to increase the production of riboflavin while
maintaining the production of L-lysine or L-threonine at a high
15 level. Particularly, a mutant microorganism was produced by
modifying KCCM11222P as a parent strain to increase the
production of riboflavin while maintaining the production of Lthreonine
at a high level.
The Corynebacterium sp. microorganism having the ability
20 of producing L-lysine may be a microorganism that produces Llysine
with increased efficiency. Methods for increasing the
production efficiency of L-lysine include a method of
amplifying a gene that is involved in the L-lysine biosynthesis
pathway or modifying the promoter of the gene to increase the
25 enzymatic activity. Examples of enzymes involved in L-lysine
15
biosynthesis include aspartate aminotransferase, aspartokinase,
aspartate semialdehyde dehydrogenase, pyruvate carboxylase,
dihydrodipicolate reductase, dihydrodipicolinate synthase,
diaminopimelate decarboxylase, and the like. Previous patent
5 related to Coryne-type bacterium-derived promoters includes
WO09/096690 relating to the improved promoter of
dihydrodipicolinate reductase, and Korean Granted Patent No.
0930203 discloses the improved promoters of aspartokinase and
aspartate semialdehyde dehydrogenase. Also, Korean Granted
10 Patent No. 0924065 discloses a method of improving the
production of L-lysine by introducing one or more copies of the
above-mentioned biosynthesis-related genes and replacing the
promoters of the genes with exogenous strong promoters.
The Corynebacterium sp. microorganism having the ability
15 to produce L-threonine may be a microorganism that produces Lthreonine
with increased efficiency. In order to improve the
ability to produce L-threonine, conventional methods can be
used by those skilled in the art such as not only the
acquisition of auxotrophic mutants, analogue resistant mutants ,
20 metabolism regulatory mutants, but also the construction of
recombinant strains having increased activity of L-threonine
biosynthetic enzyme. For example, a mutant strain or a
recombinant strain can be modified so that the L-threonine
biosynthetic enzyme does not undergo feedback inhibition, or a
25 recombinant strain can be modified to increase the expression
16
of L-threonine biosynthetic enzyme gene. In the modification
of the L-threonine-producing strain by these methods,
properties such as auxotrophy, analogue resistance and
metabolism regulatory mutations can be provided alone or in
5 combination. If the activity of L-threonine biosynthetic
enzyme is enhanced, the activity of one or more of these
enzymes can be enhanced. Examples of the gene encoding Lthreonine
biosynthetic enzyme include aspartokinase III gene
(lysC), aspartate semialdehyde dehydrogenase gene (asd),
10 aspartokinase I gene (thrA), homoserine kinase gene (thrB) and
threonine synthase gene (thrC) which are included in the thr
operon. The activity of L-threonine biosynthetic enzyme can be
increased by introducing a mutation into the gene encoding the
enzyme or amplifying the gene to increase the intracellular
15 activity of the enzyme. These can be achieved using genetic
recombination technology. Also, the production of L-threonine
can be increased by deleting L-threonine dehydrogenase activity
related to threonine degradation. In addition to the enzymes
of the L-threonine degradation system, the production of L20
threonine can be increased by reducing or deleting enzymes that
are involved in the glycolytic pathway, the TCA cycle or the
respiratory chain process, which adversely affect the
production of L-threonine, enzymes that regulate the expression
of genes, or the enzymes of the byproduct biosynthetic system.
25 Examples of a method for improving the efficiency of production
17
of L-threonine include a method of modifying a microorganism to
enhance the expression of enzymes that are involved in the Lthreonine
biosynthesis pathway. Enzymes that are involved in
L-threonine biosynthesis include homoserine dehydrogenase,
5 homoserine kinase, threonine synthase, and threonine exporter.
Other examples of a method for imparting or enhancing the
ability to produce L-threonine include a method of introducing
a mutation to make homoserine dehydrogenase resistant to
feedback by threonine (US Patent No. 6,649,379). In a specific
10 example of the present invention, mutant microorganism
KFCC10881-THR (accession number: KCCM11222P) was produced by
modifying KFCC10881 to have resistance to the L-threonine
analogue AHV (2-amino-3-hydroxy-valerate).
Specifically, the modified Corynebacterium sp.
15 microorganism may be a modified Corynebacterium sp.
microorganism for producing high concentrations of L-amino acid
and riboflavin, which is obtained by modifying a
Corynebacterium sp. microorganism having high L-amino acidproducing
ability so as to increase the production of
20 riboflavin while maintaining the production of L-amino acid at
a high level, as a result of enhancing the activity of enzymes
that are expressed by the rib operon. Furthermore, it may be a
microorganism obtained by enhancing the activity of the enzyme
that is expressed by the riboflavin biosynthesis gene family of
25 the rib operon.
18
The method of enhancing the activity of the enzyme may be,
for example, one or more selected from the group consisting of
a method of increasing the intracellular copy number of a gene
encoding each enzyme of the “operon or enzyme family”
5 (hereinafter referred to as “enzyme family”), a method of
introducing a mutation into an expression regulatory sequence
for the chromosomal gene encoding each enzyme of the enzyme
family, a method of replacing the expression regulatory
sequence for the chromosomal gene encoding each enzyme of the
10 enzyme family with a sequence having strong activity, a method
of substituting the chromosomal gene encoding the enzyme with a
gene mutated to increase the activity of the enzyme family, and
a method of introducing a mutation into the chromosomal gene
encoding each enzyme of the enzyme family to enhance the
15 activity of the gene family, but is not limited thereto. This
method can be performed by various methods known in the art.
Specifically, the method may be a method of replacing the
regulatory sequence for the chromosomal gene encoding the
enzyme family, which is expressed by the rib operon, with a
20 strong promoter, a method of inserting a strong promoter to
upstream of the chromosomal gene encoding RibG (pyrimidine
deaminase-reductase) located in the front of the riboflavin
biosynthesis gene family, or a method of increasing the
intracellular copy number of the gene encoding either the
25 enzyme family that are expressed by the rib operon or the
19
enzyme family that is expressed by the riboflavin biosynthesis
gene family.
The expression “increasing the intracellular copy number”
may include the case in which the enzyme-encoding gene is
5 operably linked into the chromosome to stably express the
enzyme or the case in which a vector comprising the enzymeencoding
gene is operably transformed into the chromosome to
stably express the enzyme. As used herein, the term “vector”
refers to a DNA construct containing the nucleotide sequence of
10 a gene operably linked to a suitable regulatory sequence so as
to express the target gene in a suitable host cell.
Based on the results obtained by increasing the copy
number of the gene encoding the enzyme that is expressed by rib
operon in the chromosomal DNA, any person skilled in the art
15 could appreciate that an increase in the copy number of the
gene encoding the enzyme that is expressed by the rib operon
outside the chromosome by a vector, a modification of the
regulatory region of the gene encoding the enzyme that is
expressed by the rib operon inside or outside the chromosome or
20 a modification of the gene itself to increase expression would
achieve the same result.
When a vector is used, it is possible to prepare a
microorganism having enhanced activity of the enzyme that is
expressed by the rib operon, by transforming a Corynebacterium
25 sp. microorganism having L-amino acid-producing ability with a
20
recombinant vector having a nucleotide sequence introduced
therein. The vector that may be used in the present invention
is not specifically limited, and a known vector may be used in
the present invention. Examples of vectors that may be used in
5 the present invention include pACYC177, pACYC184, pCL, pECCG117,
pUC19, pBR322 and pMW118 vectors. In a specific example of the
present invention, pECCG117 was used.
In prokaryotes, the regulatory sequence includes a
promoter capable of initiating transcription, any operator for
10 regulating this transcription, a suitable ribosome binding site
(RBS) encoding a suitable mRNA ribosome binding site, a
sequence for regulating the termination of transcription, and a
sequence for regulating the termination of translation.
15 In place of the inherent promoter located upstream of the
gene encoding the enzyme, an improved promoter having a
nucleotide substitution mutation, generated from the inherent
promoter or heterogeneous promoter, can be used. Examples of
the heterogeneous promoter include pcj7 promoter, lysCP1
20 promoter, EF-Tu promoter, groEL promoter, aceA promoter, aceB
promoter and the like. Specifically, pcj7 promoter or lysCP1
promoter from Corynebacterium may be used. Most specifically,
lysCP1 promoter may be used.
As used herein, the term "lysCP1 promoter" means a
25 promoter improved by nucleotide substitution of a promoter
21
region of the gene encoding aspartate kinase and aspartate
semialdehyde dehydrogenase, and a strong promoter that
increases the expression level of aspartate kinase gene to
improve the enzymatic activity to be approximately 5 times more
5 than that of the wild-type (WO 2009/096689). The lysCP1
promoter can be nucleotide residues 1 to 353 (SEQ ID NO: 6) of
SEQ ID NO: 17, which comprise the lysCP1 promoter sequence and
the part of lysC-asd gene. Specifically, the vector was
constructed such that an improved promoter of SEQ ID NO: 5
10 comprising a nucleotide substitution mutation or the improved
promoter of lyc C gene, lysCP1 of SEQ ID NO: 6, having strong
expression-inducing activity can be introduced into the
chromosome of the host cell. Methods for overexpressing the
target gene include a method of improving a promoter by
15 substituting some nucleotides of the inherent promoter sequence
with other nucleotides to increase the expression level, or a
method of substituting a promoter with the promoter of another
gene having a high expression level (WO 2009/096689).
As used herein, the term "transformation" means an
20 overall action of introducing a gene into the host cell,
Corynebacterium sp. for its expression in the host cell. In
this regard, the promoter and the gene are polynucleotides,
including DNA and RNA. As long as the gene can be introduced
in the host cell and expressed therein, any type of the gene
25 can be used. For example, the gene can be introduced into the
22
host cell in a form of an expression cassette which is a
polynucleotide construct including all elements for expressing
the gene. The expression cassette includes a promoter which is
operably linked to the gene, a transcription termination signal,
5 a RBS, and a translation termination signal. The expression
cassette may be a form of an expression vector capable of selfreplication.
The gene also may be introduced into the host
cell by itself or in a polynucleotide construct to be operably
linked to the sequence necessary for expression in the host
10 cell.
Methods for transforming the vector of the present
invention include any method for introducing nucleic acid into
a cell, and a suitable standard transformation technique known
in the art can be selected depending on a host cell. Examples
15 of the transformation method include electroporation, calcium
phosphate (CaPO4) precipitation, calcium chloride (CaCl2)
precipitation, microinjection, a polyethylene glycol (PEG)
method, a DEAE-dextran method, a cation liposome method, a
lithium acetate-DMSO method and the like.
20 The host cell in the present invention can be a host in
which DNA is introduced with high efficiency and expressed with
high efficiency. Specifically, the Corynebacterium sp.
microorganism as described above may be used. More
specifically, Corynebacterium glutamicum, particularly
25 Corynebacterium glutamicum KFCC10881, may be used. As the
23
microorganism having L-threonine-producing ability, any
microorganism capable of producing L-threonine can be used
without limitation. Specifically, KFCC10881-THR (accession
number: KCCM11222P) may be used.
5 Specifically, the microorganism can be obtained by
replacing the promoter located in the upstream of the
chromosomal region, which encodes the enzyme family that is
expressed by the rib operon, with the promoter of SEQ ID NO: 5,
or replacing the upstream promoter of ribG gene with the
10 promoter of SEQ ID NO: 6. In a specific example of the present
invention, the promoter of KFCC10881 strain was replaced with
the promoter of SEQ ID NO: 5, and as a result, it was shown
that the average concentration of L-lysine did not change, but
the average concentration of riboflavin increased by about 61
15 times (Table 2). Also, it was found that, when the upstream
promoter of the ribG gene was replaced with the promoter of SEQ
ID NO: 6, the average concentration of riboflavin increased by
about 73 times (Table 3), and when the copy number of the rib
operon was increased, the average concentration of riboflavin
20 increased by 66 times (Table 4). In addition, it was shown
that, when the promoter of L-threonine-producing strain
KFCC10881-THR was replaced with the promoter of SEQ ID NO: 5 or
6, the average concentration of L-threonine rarely changed, but
the concentrations of riboflavin increased by 56 times and 66
25 times, respectively (Table 6).
24
Specifically, a Corynebacterium sp. microorganism having
the ability to produce L-lysine or L-threonine can be modified
to produce a high concentration of riboflavin together with a
high concentration of L-lysine or L-threonine, by inserting a
5 strong promoter in the front of the gene encoding Rpe
(ribulose-5-phosphate-3-epimerase) located upstream of the rib
operon, or inserting a strong promoter into upstream of the
gene encoding the RibG (pyrimidine deaminase-reductase) located
in the front of the riboflavin biosynthesis gene family (FIG.
10 2), to enhancing of the activity of the enzyme family encoded
by rib operon or riboflavin biosynthesis gene family. In a
specific example of the present invention, it was found that
there was a mutation in a nucleotide sequence of -10th to -16th
nucleotide residues of the nucleotide sequence of rpe located
15 upstream of rib operon of the KCCM11223P strain. Thus, the
expected promoter region (SEQ ID NO: 5) of the rib operon of
the KCCM11223P strain was cloned and then introduced into the
chromosome of a parent strain having the ability to produce Llysine,
and as a result, it was found that the production of
20 riboflavin in the strain increased to a similar level to that
in the KCCM11223P strain while the production of L-lysine was
maintained (Table 2). Accordingly, it was predicted that the
production of riboflavin would be increased by the enhancement
of the rib operon, and a strong promoter was introduced to
25 upstream of ribG located in the front of the riboflavin
25
biosynthesis gene family in the rib operon, and as a result, it
was shown that the production of riboflavin in the modified
strain increased by about 73 times compared to that in the
parent strain (Table 3). In addition, it was found that, even
5 when the copy number of the rib operon was increased, the
production of riboflavin increased to a level similar to that
in the use of the strong promoter (Table 4). Furthermore, when
the promoter upstream of rpe in the parent strain having Lthreonine-
producing ability was replaced with the promoter of
10 SEQ ID NO: 5 according to the present invention and when a
strong promoter was introduced to upstream of ribG, it was
found that the production of riboflavin increased by about 60-
70 times while the production of L-threonine was maintained at
a level similar to that in the parent strain (Table 6). Thus,
15 it was found that the inventive Corynebacterium sp.
microorganism obtained by modifying the parent strain to
increase the production of riboflavin while maintaining the
production of amino acid at a high level can produce high
concentrations of L-amino acid and riboflavin at the same time.
20
Specifically, the modified Corynebacterium sp.
microorganism that simultaneously produces L-lysine and
riboflavin may be a Corynebacterium glutamicum (accession
number: KCCM11223P) modified by inducing a random mutation in a
25 Corynebacterium glutamicum having high L-lysine-producing
26
ability so as to increase the production of riboflavin while
maintaining the production of L-lysine at a high level.
Furthermore, it may be a modified Corynebacterium glutamicum
(accession number: KCCM11220P, KCCM11221P or KCCM11223P)
5 obtained by modifying the Corynebacterium sp. microorganism
having L-lysine-producing ability so as to increase the
production of riboflavin while maintaining the production of Llysine
at a high level, as a result of enhancing the activity
of the enzyme family that is expressed by the rib operon
10 comprising the riboflavin biosynthesis gene family.
Performing random mutagenesis to increase carbon flow in
the pentose-phosphate pathway of the L-lysine-producing strain,
the present inventors found a microbial colony having a deep
yellow color, and have found that the production of riboflavin
15 in the mutant microorganism increased by about 60 times while
the production of L-lysine was maintained at a level similar to
that in the parent strain (Table 1). The present inventors
have determined that this characteristic of the mutant strain
is useful in the production of feed additives that should
20 contain amino acid and riboflavin at a constant ratio.
Accordingly, the mutant strain according to the present
invention was named “Corynebacterium glutamicum CA01-2183” or
“KFCC10881-YC” and deposited with the Korean Culture Center of
Microorganisms (Yurim B/D, Honje 1-dong, Sudaemun-gu, Seoul,
25 Korea) on November 11, 2011 under the accession number
27
KCCM11223P. In addition, the mutant strain obtained by
introducing the Pmrb promoter into the rib operon promoter of
the parent strain KFCC10881 was named “Corynebacterium
glutamicum CA01-2162” or “KFCC10881::Pmrb” and deposited with
5 the Korean Culture Center of Microorganisms (Yurim B/D, Honje
1-dong, Sudaemun-gu, Seoul, Korea) on November 11, 2011 under
the accession number KCCM11221P. In addition, the strain
obtained by introducing lysCP1 upstream of the initiation codon
of the ribG gene of the parent strain KFCC10881 was named
10 “Corynebacterium glutamicum CA01-2161” or
“KFCC10881::lysCP1_ribG”. It was deposited with the Korean
Culture Center of Microorganisms (Yurim B/D, Honje 1-dong,
Sudaemun-gu, Seoul, Korea) on November 11, 2011 under the
accession number KCCM11220P.
15 The ratio of L-amino acid and riboflavin in the fermented
culture medium, simultaneously produced by the method of the
present invention, is 1:0.0001-0.1. If the L-amino acid is Llysine,
the ratio of L-lysine and riboflavin is specifically
1:0.001-0.05, and more specifically 1:0.003-0.01. If the L20
amino acid is L-threonine, the ratio of L-threonine and
riboflavin is specifically 1:0.001-0.1, and more specifically
1:0.005-0.05.
Culture of the Corynebacterium sp. microorganism can be
performed in a suitable medium by various culture methods known
25 in the art (Chmiel, Bipprozesstechnik 1. Einfuhrung in die
28
Bioverfahrenstechnik ,Gustav Fischer Verlag, Stuttgart, 1991;
Storhas, Bioreaktoren und periphere Einrichtungen, Vieweg
Verlag, Braunschweig/Wiesbaden, 1994). Examples of the culture
method include batch culture, fed-batch culture, and continuous
5 culture. Examples of the fed-batch culture include fed-batch
culture and repeated fed-batch culture, but are not limited
thereto.
In addition, a medium that may be used in culture in the
present invention may be a suitable medium known in the art
10 depending on the culture method and strain selected ("Manual of
Methods for General Bacteriology" by the American Society for
Bacteriology, Washington D.C., USA, 1981). The medium that is
used in the present invention may contain various carbon
sources, nitrogen sources and trace elements. The medium for
15 culture of Corynebacterium microorganisms may contain, as
carbon sources, sucrose, glucose, fructose, fat, fatty acid,
alcohol, organic acid and the like. Specific examples of
carbon sources that may be used in the present inventions
include carbohydrates such as molasses, glucose, lactose,
20 fructose, maltose, starch and cellulose, oils and fats such as
soybean oil, sunflower oil, castor oil and coconut oil, fatty
acids such as palmitic acid, stearic acid and linoleic acid,
alcohols such as glycerol and ethanol, and organic acids such
as acetic acid. These carbon sources can be used in a suitable
25 amount. Examples of nitrogen sources that may be used in the
29
present invention include organic nitrogen sources such as
peptone, yeast extract, meat juice, malt extract, corn steep
liquor, and soybean cake hydrolysates, and inorganic nitrogen
sources such as urea, ammonium sulfate, ammonium chloride,
5 ammonium phosphate, ammonium carbonate and ammonium nitrate.
These nitrogen sources may be used alone or in combination.
The medium may contain, as phosphorus sources, potassium
phosphate monobasic, potassium phosphate dibasic and
corresponding sodium-containing salts. The medium may contain
10 metal salts such as magnesium sulfate or iron sulfate. In
addition, the medium may contain amino acids, vitamins and
suitable precursors. These sources or precursors may be added
to the medium in a batch or continuous manner.
Compounds such as ammonium hydroxide, potassium
15 hydroxide, ammonia, phosphoric acid and sulfuric acid may be
added to the medium in a suitable manner during culturing to
adjust the pH of the culture medium. In addition, during
culturing, a defoaming agent such as fatty acid polyglycol
ester may be used to suppress the formation of foam. Further,
20 in order to maintain the culture medium in an aerobic state,
oxygen or oxygen-containing gas (e.g., air) can be injected
into the culture medium. The culture medium can typically be
maintained at a temperature ranging from 20 ℃ to 45 ℃, and
specifically from 25 ℃ to 40 ℃. As for the culture period,
25 culture can be continued until the desired level of L-amino
30
acid will be obtained. Specifically, the culture period can be
10-160 hours.
The method of the present invention may further comprise
a step of granulating the fermented culture medium comprising
5 L-amino acid and riboflavin. The fermented culture medium may
contain a bacterial sludge or may be free of the bacterial
sludge. To remove the bacterial sludge, the method may further
comprise a step of removing the bacterial sludge from the
fermented culture medium containing L-threonine and riboflavin.
10 If the fermented culture medium contains bacterial sludge,
a granular formulation can be produced, which does not require
a filtration operation for removing the bacterial sludge and
has a low moisture-absorbing property even when a moisture
absorption preventing agent is not added thereto. Also, the
15 granular formulation has good flowability and high apparent
density, and the amino acid content thereof can be controlled.
The granulation method can be performed using the method
described in, for example, Korean Patent Registration No.
0838200 or No. 0338578. Specifically, the granulation method
20 may comprise the step of: concentrating the fermentation broth;
adding an excipient to the concentrate to form a mixed
concentrate; introducing particulate seeds having a size of
200-500 ㎛ into a granulation machine; and spraying the mixed
concentrate from the bottom of the granulation machine while
25 adding hot air to coat the particulate seeds with the
31
concentrate while forming granules, but is not limited thereto.
If the fermentation broth contains no bacterial sludge,
the granulation method may comprise the step of: filtering the
fermented culture medium to remove the bacterial sludge;
5 concentrating the filtrate; drying the concentrate to form
granules; and coating the granules with a coating agent such as
an excipient or a moisture absorption-preventing agent, but is
not limited thereto.
The bacterial sludge can be removed from the fermented
10 culture medium by separating L-amino acid and riboflavin using
methods such as centrifugation, filtration, ion-exchange
chromatography and crystallization. Specifically, L-amino
acid and riboflavin can be separated by centrifuging the
fermented culture medium at low speed to remove the bacterial
15 sludge and separating the supernatant by ion exchange
chromatography, but are not limited thereto.
In another aspect, the present invention provides a
modified Corynebacterium glutamicum microorganism which is
20 obtained by inducing a random mutation in a Corynebacterium
glutamicum microorganism having high-concentration L-lysineproducing
ability so as to increase the production of
riboflavin while maintaining the production of L-lysine at a
high level, wherein the modified Corynebacterium glutamicum
25 microorganism is deposited under accession No. KCCM11223P.
32
Herein, the microorganism is as described above.
In still another aspect, the present invention provides a
modified Corynebacterium sp. microorganism for producing L-
5 lysine or L-threonine, and riboflavin, which is obtained by
enhancing activity of an enzyme family that is expressed by the
rib operon comprising riboflavin biosynthesis gene family in a
Corynebacterium sp. microorganism having L-lysine or Lthreonine-
producing ability so as to increase the production of
10 riboflavin while maintaining the production of L-lysine or Lthreonine
at a high level.
Herein, the microorganism, the enhancement of the enzyme
family, and the modified Corynebacterium sp. microorganism are
as described above.
15
In still another aspect, the present invention provides a
granular formulation prepared by a method comprising: a)
culturing a microorganism which is obtained by modifying a
Corynebacterium sp. microorganism having L-lysine or L20
threonine-producing ability so as to increase the production of
riboflavin while maintaining the production of L-lysine or Lthreonine
at a high level, and producing L-lysine or Lthreonine,
and riboflavin by a fermentation process; and b)
granulating the fermented culture medium of step a), which
25 comprises L-lysine or L-threonine, and riboflavin.
33
Specifically, the L-amino acid may be L-lysine or Lthreonine.
The modified microorganism is as described above.
The method for preparing the granular formulation may
further comprise a step of removing a bacterial sludge from the
5 fermented culture medium of step a), which comprises L-lysine
or L-threonine, and riboflavin. In other words, as described
above, the granular formulation of the present invention may
contain or not contain the bacterial sludge.
Specifically, the ratio of L-lysine or L-threonine:
10 riboflavin in the granular formulation may be 1:0.0001-0.01 as
described above.
As used herein, the term “granular formulation” means a
granule-type formulation that overcomes the disadvantages of
conventional power formulations, such as dust generation and
15 product loss.
In still another aspect, the present invention provides a
formulation comprising L-lysine or L-threonine, and riboflavin,
the formulation is prepared by a method comprising: a)
20 culturing a microorganism which is obtained by modifying a
Corynebacterium sp. microorganism having L-lysine or Lthreonine-
producing ability so as to increase the production of
riboflavin while maintaining the production of L-lysine or Lthreonine
at a high level, and producing L-lysine or L25
threonine, and riboflavin by a fermentation process; and
34
b) removing a bacterial sludge from the fermented culture
medium of step a), which comprises L-lysine or L-threonine, and
riboflavin.
Specifically, the ratio of L-lysine or L-threonine:
5 riboflavin in the formulation may be 1:0.0001-0.01 as described
above.
In still another aspect, the present invention provides a
feed or a feed additive, which comprises the above-described
10 granular formulation or the formulation comprising L-lysine, Lthreonine
and riboflavin.
In high concentrations of L-lysine or L-threonine, and
riboflavin produced in a specific example of the present
invention, the ratio of lysine: riboflavin was about 1:0.005,
15 or the ratio of L-threonine: riboflavin was about 1:0.03 (see
Tables 1 to 6). The requirements of components in animal feed
are about 1-5 g/kg for L-lysine, about 0.6-3.3 g/kg for Lthreonine,
and 2-4 mg/kg for riboflavin, which is about 0.1% of
the requirement of L-lysine (NRC. 1998. National Academy of
20 Sciences- National Research Council, Washington, D.C.), and
toxicity by administration of large amounts of L-lysine or Lthreonine
and riboflavin was not reported. Thus, it was found
that L-amino acid and riboflavin, simultaneously produced by
the method of the present invention are suitable for use as
25 feed additives.
35
Specifically, the L-amino acid may be L-lysine or Lthreonine.
Specifically, the ratio of L-lysine or L-threonine:
riboflavin in the feed or feed additive may be 1:0.0001-0.01 as
5 described above.
The feed of the present invention can be prepared by
preparing a feed additive comprising the L-amino acid and
riboflavin, and mixing the feed additive with feed or adding
10 the feed additive directly to feed during the preparation of
the feed. The L-amino and riboflavin in the feed of the
present invention may be in a liquid or dry state, and
specifically in the form of dry powder. Examples of a drying
method that may be used in the present invention include, but
15 are not limited to, air drying, natural drying, spray drying
and freeze drying. The livestock feed may comprise, in
addition to the L-amino acid and riboflavin of the present
invention, conventional additives capable of enhancing the
preservative property of the feed.
20 The feed additive of the present invention may further
comprise non-pathogenic other microorganisms. Microorganisms
that may be added to the feed additive of the present invention
may be selected from the group consisting of Bacillus subtilis
capable of producing protease, lipase and glucose-converting
25 enzyme, Lactobacillus sp. that has physiological activity and
36
organism-degrading ability in anaerobic conditions such as
cow’s stomach, filamentous fungi such as Aspergillus oryzae
that shows the effects of increasing the weight of livestock,
the production of milk and the digestion/absorption of the feed
5 (J AnimalSci 43: 910-926, 1976), and yeast such as
Saccharomyces cerevisiae (J Anim Sci 56:735-739, 1983).
Examples of feed containing the L-amino acid and
riboflavin include, but are not limited to; vegetable feeds,
such as grains, roots/fruits, food processing byproducts, algae,
10 fibers, pharmaceutical byproducts, oils and fats, starches,
gourds, and grain byproducts, and animal feeds such as proteins,
inorganic materials, oils and fats, minerals, single-cell
proteins, animal planktons, and food residue.
Feed additives comprising the L-amino acid and riboflavin
15 of the present invention include, but are not limited to; a
binder, an emulsifier and a preservative, which are added to
prevent deterioration in quality, amino acids, vitamins,
enzymes, probiotics, flavoring agents, non-protein nitrogenous
compounds, silicates, buffer, colorants, extracting agents, and
20 oligosaccharides, which are added to increase effects, and
other feed mixture.
Mode for Invention
Hereinafter, the present invention will be described in
25 further detail with reference to examples. It is to be
37
understood, however, that these examples are for illustrative
purposes only and are not intended to limit the scope of the
present invention.
5 Example 1: Screening of gluconic acid tolerant strain by
artificial mutagenesis
In this Example, in order to increase carbon flow in the
pentose-phosphate pathway that is the major source of
nicotinamide adenine dinucleotide phosphate (NADPH) required
10 for the production of L-amino acid, an experiment for imparting
adaptability for high-concentration of gluconic acid to the
Corynebacterium glutamicum strain was performed.
An artificial mutation in KFCC10881 (Korean Patent No.
0159812), as a parent strain, was induced by N-methyl-N-nitro-
15 N-nitroguanidine (NTG) and then the strain was cultured in the
following plate medium containing 20 g/ℓ of gluconic acid while
strains that formed colonies faster than a control group not
treated with NTG were isolated. In the case of the control
group, the average diameter of colonies formed at 60 hours was
20 about 0.5 mm, whereas in the case of the NTG-treated group, a
diameter of colonies reached 1 mm within 40 hours of culture.
Among the colonies, a colony having a deep yellow color unlike
the control group was found, and thus it was expected that a
mutant having a new character would be produced. This strain
25 was named “Corynebacterium glutamicum CA01-2183”, or
38
“KFCC10881-YC”, and was examined the cause of color
development. The mutant strain, KFCC10881-YC, was deposited
with the Korean Culture Center of Microorganisms (Yurim B/D,
Honje 1-dong, Sudaemun-gu, Seoul, Korea) on November 11, 2011
5 under the accession number KCCM11223P.
Composition of plate medium (pH 7.0)
20 g gluconic acid, 50 g (NH4)2SO4, 10 g peptone, 5 g yeast
extract, 1.5 g urea, 5 g KH2PO4, 10 g K2HPO4, 0.5 g MgSO4•7H2O,
10 100 ㎍ biotin, 1000 ㎍ thiamine chloride, 2000 ㎍ calciumpantothenic
acid, 2000 ㎍ nicotinamide, and 20 g agar (per liter
of distilled water).
Example 2: Analysis of the Lysine-producing ability of
15 KFCC10881-YC and examination of cause of color development
In order to examine the characteristics of the KFCC10881-
YC strain prepared in Example 1 above, the strain was cultured
in the following manner to compare the Lysine-producing ability
thereof, and the components of the culture medium were analyzed.
20 Specifically, each strain was inoculated into a 250-㎖
corner-baffle flask containing 25 ㎖ of seed medium, and then
cultured at 30 ℃ for 20 hours with shaking at 200 rpm. Next, 1
㎖ of the seed culture was inoculated into a 250-㎖ cornerbaffle
flask containing 24 ㎖ of production medium and cultured
25 at 30 ℃ for 72 hours with shaking at 200 rpm. The compositions
39
of the seed medium and the production medium are as follows.
Composition of seed medium (pH 7.0)
20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea,
5 4 g KH2PO4, 8g K2HPO4, 0.5 g MgSO4 7H2O, 100 ㎍ biotin, 1000 ㎍
thiamine HCl, 2000 ㎍ calcium- pantothenic acid, and 2000 ㎍
nicotinamide (per liter of distilled water)
Composition of production medium (pH 7.0)
10 100 g glucose, 40 g (NH4)2SO4, 2.5 g soybean extract, 5 g
corn steep solids, 3 g urea, 1 g KH2PO4, 0.5 g MgSO4•7H2O, 100 ㎍
biotin, 1000 ㎍ thiamine HCl, 2000 ㎍ calcium- pantothenic acid,
3000 ㎍ nicotinamide, and 30 g CaCO3 (per liter of distilled
water).
15
After completion of the culture, the KFCC10881-YC strain
maintained the deep yellow color, and the culture supernatant
also had a deep yellow color, unlike the culture medium of the
control KFCC10881. Accordingly, the present inventors assumed
20 that the material causing this color development would be
contained in the culture medium. To verify this assumption,
the concentrations of carotinoids and riboflavin that influence
the color development in the microorganism were analyzed. As a
result, the concentration of riboflavin in the culture medium
25 showed a clear difference from that in the control, and there
40
was no significant difference in the concentration of
carotenoids. The concentrations of L-lysine and riboflavin
analyzed by HPLC were shown in Table 1 below.
5 Table 1: Concentrations of L-lysine and riboflavin
produced in KFCC10881-YC
Test
group
Strain L-lysine (g/ℓ) Riboflavin (mg/ℓ)
Batch
1
Batch
2
Batch
3
Average Batch
1
Batch
2
Batch
3
Average
1 KFCC10881 43.1 43.5 44.1 43.5 3.4 3.5 3.0 3.3
2 KFCC10881-
YC
43.7 44.3 42.8 43.6 191.2 187.5 189.0 189.2
As a result, as seen from the Table 1, the average
concentration of L-lysine in KFCC10881-YC was similar to that
10 in the L-lysine-producing strain KFCC10881, but the average
concentration of riboflavin in KFCC10881-YC increased by 57
times. Thus, it could be seen that the deep yellow color of
the colony, which is the distinct characteristic distinguished
from the parent strain, is attributable to the increased in
15 concentration of riboflavin.
Example 3: Examination of modification in promoter
upstream of rib operon of strain producing high concentration
of riboflavin, and construction of a vector for chromosomal
20 integration of the modified promoter (Pmrb)
41
In order to investigate the nucleotide mutation that
induced the color change and increased the riboflavin producing
ability in the KFCC10881-YC strain prepared in Example 1,
nucleotide sequence of the chromosomal region related to the
5 riboflavin biosynthesis in the Corynebacterium glutamicum sp.
KFCC10881-YC strain was determined and confirmed based on the
NIH GenBank database.
As a result, it was found that -10th and -16th nucleotide
residues from the initiation codon of the rpe gene located
10 upstream of the rib operon had a G-to-A substitution and a Gto-
T substitution, respectively. The promoter having the two
nucleotide substitution in the expected region of the rib
operon promoter was named “Pmrb” (SEQ ID NO: 5).
In order to examine the effect of increase in riboflavin
15 concentration by increases in the expression inducing activity
and enzymatic activity of the Pmrb promoter having the
nucleotide substitutions , a vector for introducing the
promoter into the chromosome was constructed.
Based on the reported nucleotide sequence, a primer
20 having an EcoRI restriction enzyme site inserted into the 5’
end and a primer having a SalI restriction enzyme site inserted
into the 3’ end (SEQ ID NOS: 7 and 8) were synthesized. Using
the promoters, PCR was performed with the chromosome of
KFCC10881-YC as a template to amplify an about 650-bp Pm-rpe
25 gene fragment including a 350-bp region expected to be the rib
42
operon promoter. The PCR was performed under the following
conditions: initial denaturation at 94 ℃ for 5 minutes, and
then 30 cycles, each consisting of denaturation at 94 ℃ for 30
sec, annealing at 56 ℃ for 30 sec, and extension at 72 ℃ for
5 40 sec, followed by final extension at 72 ℃ for 7 min. The
primers used in the PCR are as follows.
SEQ ID NO. 7: 5'-tttgaattcgtgtgcgtgcaggtttctc-3'
SEQ ID NO. 8: 5'-tttgtcgacattccgctaaaacacgt-3'
The gene fragment amplified by PCR was treated with the
10 restriction enzymes EcoRI and SalI to obtain a DNA fragment,
which was then ligated with a pDZ vector (Korean Patent
Publication No. 2009-0094433), which has the EcoRI and SalI
sites at the ends, for introduction into the chromosome. Then,
the vector was transformed into E. coli DH5α, which was then
15 plated on LB solid medium containing kanamycin (25 mg/ℓ). A
colony transformed with the vector comprising the desired gene
was screened by PCR, and then a plasmid was obtained using a
conventional plasmid extraction method known in the art. This
plasmid was named “pDZ-Pmrb” (FIG. 3).
20
Example 4: Construction of a strain having Pmrb
introduced into upstream of rib operon in chromosome of highconcentration
lysine-producing strain and comparison of lysine
and riboflavin productivities
25 The vector pDZ-Pmrb prepared in Example 3 was transformed
43
into the L-lysine-producing strain Corynebacterium glutamicum
KFCC10881 by homologous chromosomal recombination. Then, a
strain having a rib operon promoter mutation introduced into
the chromosome was separated based on a change in the color of
5 colonies and cultured in the same manner as described in
Example 2, and the concentrations of L-lysine and riboflavin
recovered from the culture were analyzed (Table 2). The strain,
the rib operon promoter mutation introduced therein, was named
“Corynebacterium glutamicum CA01-2162” or “KFCC10881::Pmrb”.
10 The mutant strain, KFCC10881::Pmrb, was deposited with the
Korean Culture Center of Microorganisms (Yurim B/D, Honje 1-
dong, Sudaemun-gu, Seoul, Korea) on November 11, 2011 under the
accession number KCCM11221P.
Table 2: Anaylsis of production of L-lysine and
15 riboflavin by introduction of promoter mutation
Test
group
Strain L-lysine (g/l) Riboflavin (mg/l)
Batch
1
Batch
2
Batch
3
Average Batch
1
Batch
2
Batch
3
Average
3 KFCC10881 42.1 42.6 43.1 42.6 3.1 3.3 3.0 3.1
4 KFCC10881::Pmrb 42.7 42.3 42.8 42.6 200.2 186.3 189.9 192.1
As a result, as seen from the Table 2 above, the average
concentration of L-lysine in the case of KFCC10881::Pmrb (test
group 4) introduced with the promoter Pmrb having the two
20 nucleotide substitution mutations did not change compared to
that in the control KFCC10881 (test group 3) having the wildtype
rpe gene promoter, but the average concentration of
44
riboflavin in test group 4 increased by about 61 times.
Example 5: Construction of a vector to introduce strong
promoter (LysCP1) into upstream of ribG in rib operon
5 In order to overexpress only the riboflavin biosynthesis
group gene excluding the rpe gene, a strong promoter derived
from Corynebacterium was ligated to the initiation codon of the
ribG gene (SEQ ID NO: 4) located in the front of the rib
biosynthesis gene family, thereby constructing a recombinant
10 vector for inducing the expression of the riboflavin
biosynthesis gene family.
To obtain a ribG gene fragment derived from
Corynebacterium glutamicum, the chromosomal DNA of
Corynebacterium glutamicum KFCC10881 was prepared as a template,
15 and primers (SEQ ID NOS: 9 and 10) designed to have an EcoRI
restriction enzyme site at the 5' end and a XbaI restriction
enzyme site at the 3' end were synthesized. PCR was performed
using the synthesized primers to obtain a DNA fragment
comprising a 300-bp region upstream of the initiation codon of
20 the ribG gene. Also, primers (SEQ ID NOS: 11 and 12)
designed to have XbaI and NdeI restriction enzyme sites at the
5' end and a SalI restriction enzyme site at the 3’ end were
synthesized, and PCR was performed using the synthesized
primers to obtain a 300-bp fragment comprising the initiation
25 codon of the ribG gene. The PCR was performed under the
45
following conditions: initial denaturation at 94 ℃ for 5 min,
and then 30 cycles, each consisting of denaturation at 94 ℃ for
30 sec, annealing at 56 ℃ for 30 sec, and extension at 72 ℃
for 30 sec, followed by final extension at 72 ℃ for 7 min. The
5 two PCR products were treated with EcoRI and XbaI, and XbaI and
SalI, respectively, and then ligated with a DNA fragment
obtained by treating a pDZ vector with the restriction enzymes
SalI and EcoRI, thereby constructing a pDZ-ribG vector. The
primer sequences used in this PCR are as follows:
10 SEQ ID NO. 9: 5'- tttgaattcgtgtgcgtgcaggtttctcc-3'
SEQ ID NO. 10: 5'- tttctagattactgcgcgagtgctc-3'
SEQ ID NO. 11: 5'- ttttctagataacatatggatgttgcgcacgcg-3'
SEQ ID NO. 12: 5'- tttgtcgacattggcgtaaaacacgt-3'
15 Primers (SEQ ID NOS: 13 and 14) designed to amplify the
Corynebacterium glutamicum-derived lysCP1 promoter (SEQ ID NO:
6) and to have an XbaI restriction enzyme site inserted into
the 5' end and a NdeI restriction enzyme site inserted into the
3’ end were synthesized. Using the primers, PCR was performed
20 with the chromosomal DNA of the Corynebacterium glutamicum
strain as a template to amplify an about 400-bp promoter
fragment. The PCR was performed under the following
conditions: initial denaturation at 94 ℃ for 5 min, and then 30
cycles, each consisting of denaturation at 94 ℃ for 30 sec,
25 annealing at 58 ℃ for 30, and extension at 72 ℃ for 30 sec,
46
followed by final extension at 72 ℃ for 7 min. The PCR
amplification product was treated with XbaI and NdeI, and then
ligated with a DNA fragment obtained by treating a pDZ-ribG
vector with XbaI and NdeI, thereby constructing a pDZ-
5 lysCP1_ribG vector (FIG. 4). The primer sequences used in the
PCR are as follows:
SEQ ID NO. 13: 5'-ttttctagatagggagccatcttttgggg-3'
SEQ ID NO: 14: 5'-tttcatatgctttgtgcacctttcg-3'
10 Example 6: Comparison of lysine and riboflavin
productivities of a strain having strong promoter (LysCP1)
introduced into upstream of ribG
The vector pDZ-lysCP1_ribG prepared in Example 5 was
transformed into the L-lysine-producing strain Corynebacterium
15 glutamicum KFCC10881 by an electric pulse method. A strain
having lysCP1 substituting for the inherent promoter of the
chromosomal ribG gene was selectively isolated, thereby
obtaining KFCC10881::lysCP1_ribG that produces both L-lysine
and riboflavin. This strain was cultured in the same manner as
20 described in Example 2, and the concentrations of L-lysine and
riboflavin in the culture medium were analyzed (Table 3). The
KFCC10881::lysCP1_ribG strain was named “Corynebacterium
glutamicum CA01-2161” or “KFCC10881::lysCP1_ribG”. This mutant
strain, KFCC10881::lysCP1_ribG , was deposited with the Korean
25 Culture Center of Microorganisms (Yurim B/D, Honje 1-dong,
47
Sudaemun-gu, Seoul, Korea) on November 11, 2011 under the
accession number KCCM11220P.
Table 3: Analysis of production of L-lysine and
riboflavin by introduction of promoter
Test
group
Strain L-lysine (g/l) Riboflavin (mg/l)
Batch
1
Batch
2
Batch
3
Average Batch
1
Batch
2
Batch
3
Average
5 KFCC10881 43.7 42.5 43.1 43.1 3.4 3.5 3.0 3.3
6 KFCC10881::lysCP1_ribG 43.7 43.4 42.7 43.3 237.2 247.5 241.0 241.9
5
As a result, as shown in Table 3 above, when RibG was
overexpressed using the lysCP1 promoter (test group 6), the
average concentration of L-lysine rarely changed compared to
that in the control KFCC10881 (test group 5) having the wild10
type promoter, but the concentration of riboflavin increased by
about 73 times.
Thus, it could be seen that, when the riboflavin
biosynthesis operon was overexpressed using the heterogeneous
promoter, the production of riboflavin could be greatly
15 increased without influencing the production of lysine, and as
the expression inducing activity of the promoter became
stronger, the production of riboflavin increased by
overexpression of the riboflavin biosynthesis gene family.
20 Example 7: Comparison of lysine and riboflavin
productivities of a strain having increased copy number of rib
operon
48
A plasmid comprising the rib operon was introduced into
the KFCC10881 strain to increase the copy number of the
riboflavin biosynthesis gene, and the riboflavin production
ability of the strain was examined.
5 Based on the reported nucleotide sequence, a primer
having a XbaI restriction enzyme site inserted into the 5’ end
and a primer having the same restriction enzyme site inserted
into the 3’ end (SEQ ID NOS: 15 and 16) were synthesized, and
using the primers, PCR was performed with the chromosome of
10 KFCC10881 as a template to amplify an about 4500-bp rib operon.
The PCR was performed under the following conditions: initial
denaturation at 94 ℃ for 5 min, and then 30 cycles, each
consisting of denaturation at 94 ℃ for 30 sec, annealing at
56 ℃ for 30 sec, and extension at 72 ℃ for 4 min, followed by
15 final extension at 72 ℃ for 7 min. The primer sequences used
in the PCR are as follows:
SEQ ID NO. 15: 5'-TTTGGTACCGATTGAAAAGTCCGTGCGTG-3';
SEQ ID NO. 16: 5'-TTTGGTACCCGCGATCTTTTTCAGAAACT-3'
20
The gene fragment amplified by PCR was treated with the
restriction enzyme XbaI to obtain a DNA fragment. The DNA
fragment was ligated with a pECCG117 vector having the XbaI
restriction sites at the ends, and the vector was transformed
25 into E. coli DH5α, then plated on LB solid medium containing
49
kanamycin (25mg/ℓ). Then, a colony transformed with the vector
comprising the desired gene were screened by PCR, and then a
plasmid was obtained using a conventional plasmid extraction
method known in the art. This plasmid was named “pECCG117-rib”.
5 The vector pECCG117-rib prepared as described above was
introduced into the KFCC10881 strain by an electric pulse
method and cultured in the same manner as described in Example
2, and the concentrations of L-lysine and riboflavin in the
culture medium were analyzed (Table 4).
10
Table 4: Analysis of production of L-lysine and
riboflavin by introduction of plasmid
Test
group
Strain L-lysine (g/ℓ) Riboflavin (mg/ℓ)
Batch
1
Batch
2
Batch
3
Average Batch
1
Batch
2
Batch
3
Average
7 KFCC10881
/pECCG117
43.1 43.1 42.5 32.9 4.0 3.2 3.6 3.6
8 KFCC10881
/pECCG117-
rib
42.7 42.4 43.7 42.9 245.1 231.2 238.3 238.5
As a result, as seen in Table 4 above, in the case in
15 which the copy number of the rib operon was increased by
introduction of the plasmid (test group 8), the average
concentration of L-lysine rarely changed compared to the
control KFCC10881::pECCG117 (test group 7) containing no rib
operon, but the concentration of riboflavin increased by about
20 66 times.
50
Thus, it could be seen that, when the riboflavin
biosynthesis gene was overexpressed by increasing the copy
number of the riboflavin biosynthesis operon using the plasmid,
the production of riboflavin in the strain can be greatly
5 increased without influencing the production of the lysine.
Example 8: Construction of AHV-resistant L-threonineproducing
strain by artificial mutagenesis
In this Example, in order to increase the L-threonine10
producing ability of Corynebacterium glutamicum, an experiment
for imparting resistance to the L-threonine analogue AHV (2-
amino-3-hydroxy-valerate) was performed. Specifically, an
artificial mutation in KFCC10881 as a parent strain was induced
by N-methyl-N-nitro-N-nitroguanidine (NTG), and then the strain
15 was cultured in minimal medium containing 1-10 g/ℓ of AHV,
while whether colonies were formed at various concentrations of
AHV was analyzed in comparison with a control group not treated
with NTG. The control group not treated with NTG showed
resistance to 1-3 g/ℓ of AHV at 72 hours of culture, but in the
20 NTG-treated group, colonies were formed up to an AHV
concentration of 6 g/ℓ at the same time point. In order to
examine whether the L-threonine-producing ability of the
strains increased or not, the strains were cultured in the
following manner, and the production of L-threonine was
25 analyzed.
51
Each of the strains was inoculated into a 250-㎖ cornerbaffle
flask containing 25 ㎖ of seed medium and cultured at
30 ℃ for 20 hours with shaking at 200 rpm. Thereafter, 1 ㎖ of
the seed culture was inoculated into a 250-㎖ corner-baffle
5 flask containing 24 ㎖ of production medium and culture at 30 ℃
for 48 hours with shaking at 200 rpm. The compositions of the
seed medium and the production medium are as follows.
Composition of minimal medium (pH 7.2)
10 5 g glucose, 1 g KH2PO4, 5 g (NH4)2SO4, 0.4 g MgSO4 7H2O,
0.5 g NaCl, 200 ㎍ biotin, 100 ㎍ thiamine HCl, 100 ㎍ calciumpantothenic
acid, 0.03 g nicotinamide, 0.01 g L-threonine, 2 g
urea, 0.09 mg Na2B4O710H2O, 0.04 mg (NH4)6Mo7O274H2O, 0.01 mg
ZnSO47H2O, 0.01 mg CuSO45H2O, 0.01 mg MnCl24H2O, 1 mg FeCl36H2O,
15 and 0.01 mg CaCl2 (per liter of distilled water).
Composition of seed medium (pH 7.0)
20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea,
4 g KH2PO4, 8 g K2HPO4, 0.5 g MgSO4 7H2O, 100 ㎍ biotin, 1000 ㎍
20 thiamine HCl, 2000 ㎍ calcium- pantothenic acid, and 2000 ㎍
nicotinamide (per liter of distilled water).
Composition of production medium (pH 7.0)
100 g glucose, 20 g (NH4)2SO4, 2.5 g soybean protein, 5 g
25 corn steep solids, 3 g urea, 1 g KH2PO4, 0.5 g MgSO47H2O, 100 ㎍
52
biotin, 1000 ㎍ thiamine HCl, 2000 ㎍ calcium- pantothenic acid,
3000 ㎍ nicotinamide, and 30 g CaCO3 (per liter of distilled
water).
5 After completion of culture, the concentration of Lthreonine
in the culture medium was analyzed by HPLC, and the
mutant strain, showing the highest L-threonine production
ability, was named “Corynebacterium glutamicum CA01-2182” or
“KFCC10881-THR”. The mutant strain, KFCC10881-THR, was
10 deposited with the Korean Culture Center of Microorganisms
(Yurim B/D, Honje 1-dong, Sudaemun-gu, Seoul, Korea) on
November 11, 2011 under the accession number KCCM11222P. The
L-threonine-producing ability of KFCC10881-THR is shown in
Table 5 below.
15 Table 5: Concentration of L-threonine produced in
KFCC10881-THR
Test group Strain L-threonine (g/ℓ)
Batch 1 Batch 2 Batch 3
9 KFCC10881 1.2 1.5 1.4
10 KFCC10881-THR 6.7 7.1 7.0
In Table 5 above, test group 9 indicates the average
concentration of L-threonine in the control KFCC10881, and test
20 group 10 indicates the average concentration of L-threonine in
KFCC10881-THR.
As a result, as seen from the Table 5 above, the
53
production of L-threonine in KFCC10881-THR increased by about
5.6 g/ℓ compared to that in KFCC10881.
Example 9: Construction of strains introducingstrong
5 promoter into upstream of rib operon or upstream of ribG in
chromosome, and comparison of threonine and riboflavin
productivities
Each of the vector pDZ-Pmrb prepared in Example 3 and the
10 vector pDZ-lysCP1_ribG prepared in Example 5 was transformed
into the L-threonine-producing strain KFCC10881-THR (prepared
in Example 8) by an electric pulse method. Then, a strain
having Pmrb substituting for the wild-type promoter of the
chromosomal rpe gene, or a strain having lysCP1 substituting
15 for the wild-type promoter of the ribG gene, was selectively
isolated and was cultured in the same manner as described in
Example 7. The concentrations of L-threonine and riboflavin in
the culture medium were analyzed (Table 6).
20 Table 6: Analysis of production of L-threonine and
riboflavin by introduction of mutation into promoter
Test
group
Strain L-threonine (g/ℓ) Riboflavin (mg/ℓ)
Batch 1 Batch 2 Batch 3 Batch 1 Batch 2 Batch 3
11 KFCC10881-
THR
6.9 6.8 7.0 3.2 3.1 3.3
12 KFCC10881-
THR
7.1 6.8 7.1 180.0 177.5 179.2
54
::Pmrb
13 KFCC10881-
THR::
lysCP1_ribG
6.7 7.2 6.9 214.2 208.5 213.0
As a result, as seen from the Table 6 above, in the case
of KFCC10881::Pmrb (test group 12) comprising the promoter Pmrb
having two nucleotide substitution mutations, and KFCC10881-
5 THR::lysCP1_ribG (test group 13) having RibG overexpressed by
the lysCP1 promoter, the average concentrations of L-threonine
in the mutant strains rarely changed compared to that in the
control KFCC10881-THR (test group 11) having the wild-type
promoters of the rpe and ribG genes, but the average
10 concentrations of riboflavin in the mutant strains increased by
about 56 times and 66 times, respectively.
The above-described results indicate that, when the
riboflavin biosynthesis operon is overexpressed, the production
of riboflavin can be greatly increased without influencing the
15 production of L-threonine, thereby producing L-threonine and
riboflavin at the same time. Thus, it can be seen that, as the
expression-inducing activity of the riboflavin biosynthesis
operon promoter in the L-threonine-producing strain becomes
stronger, the production of riboflavin is increased by the
20 overexpression of the riboflavin biosynthesis gene family.
55
56
57
58
59
1. A method for producing L-lysine or L-threonine, and
riboflavin, comprising:
culturing a microorganism which is obtained by modifying a
5 Corynebacterium sp. microorganism having L-lysine or Lthreonine-
producing ability so as to increase the production of
riboflavin while maintaining the production of L-lysine or Lthreonine
at a high level; and
producing L-lysine or L-threonine, and riboflavin by a
10 fermentation process.
2. The method according to claim 1, wherein the
Corynebacterium sp. microorganism is selected from the group
consisting of Corynebacterium glutamicum, Corynebacterium
15 ammoniagenes, Corynebacterium thermoaminogenes, Brevibacterium
flavum, and Brevibacterium lactofermentum.
3. The method according to claim 2, wherein the
Corynebacterium sp. microorganism is Corynebacterium
20 glutamicum.
4. The method according to claim 1, wherein the ratio
of L-lysine or L-threonine: riboflavin is 1:0.0001-0.1.
25 5. The method according to claim 1, which further
60
comprises granulating the fermented culture medium comprising
L-lysine or L-threonine, and riboflavin.
6. The method according to claim 1, which further
5 comprises removing a bacterial sludge from the fermented
culture medium comprising L-lysine or L-threonine, and
riboflavin.
7. A modified Corynebacterium glutamicum microorganism
10 which is modified by inducing a random mutation in a
Corynebacterium glutamicum microorganism having highconcentration
L-lysine-producing ability so as to increase the
production of riboflavin while maintaining the production of Llysine
at a high level, wherein the modified Corynebacterium
15 glutamicum microorganism is deposited under accession No.
KCCM11223P.
8. A modified Corynebacterium sp. microorganism for
simultaneously producing L-lysine or L-threonine, and
20 riboflavin, which is modified by enhancing activity of an
enzyme family that is expressed by the rib operon comprising
riboflavin biosynthesis gene family in a Corynebacterium sp.
microorganism having L-lysine or L-threonine-producing ability
so as to increase the production of riboflavin while
25 maintaining the production of L-lysine or L-threonine at a high
61
level.
9. The microorganism according to claim 8, wherein the
Corynebacterium sp. microorganism is selected from the group
5 consisting of Corynebacterium glutamicum, Corynebacterium
ammoniagenes, Corynebacterium thermoaminogenes, Brevibacterium
flavum and Brevibacterium lactofermentum.
10. The microorganism according to claim 8, wherein the
10 microorganism has enhanced activity of the enzyme family that
is expressed by the riboflavin biosynthesis gene family.
11. The microorganism according to claim 10, wherein the
enhancement of the activity of the enzyme family is performed
15 by one or more selected from the group consisting of a method
of increasing the intracellular copy number of a gene encoding
each enzyme of the enzyme family, a method of introducing a
mutation into a regulatory sequence of a chromosomal gene
encoding each enzyme of the enzyme family, a method of
20 replacing the regulatory sequence of the chromosomal gene
encoding each enzyme of the enzyme family with a sequence
having strong activity, a method of substituting the
chromosomal gene encoding the enzyme with a gene mutated to
increase the activity of the enzyme family, and a method of
25 introducing a mutation into the chromosomal gene encoding each
62
enzyme of the enzyme family to enhance the activity of the gene
family.
12. The microorganism according to claim 11, wherein the
5 regulatory sequence of the chromosomal genes encoding the
enzyme family is replaced with a strong promoter.
13. The microorganism according to claim 12, wherein the
promoter is a promoter set forth in SEQ ID NO: 5 or 6.
10
14. The microorganism according to claim 8, wherein the
modified microorganism is a microorganism for simultaneously
producing L-lysine and riboflavin, which is modified by
enhancing activity of an enzyme family that is expressed by the
15 rib operon comprising the riboflavin biosynthesis gene family
in a Corynebacterium sp. microorganism having L-lysineproducing
ability so as to increase the production of
riboflavin while maintaining the production of L-lysine at a
high level, and is deposited under accession No. KCCM11220P,
20 KCCM11221P or KCCM11223P.
15. The microorganism according to claim 8, wherein the
Corynebacterium sp. microorganism having L-threonine-producing
ability is a microorganism deposited under the accession number
25 KCCM11222P.
63
16. A granular formulation prepared by a method
comprising:
a) culturing a microorganism which is obtained by
5 modifying a Corynebacterium sp. microorganism having L-lysine
or L-threonine-producing ability so as to increase the
production of riboflavin while maintaining the production of Llysine
or L-threonine at a high level, and producing L-lysine
or L-threonine, and riboflavin by a fermentation process; and
10 b) granulating the fermented culture medium of step a),
which comprises L-lysine or L-threonine, and riboflavin.
17. The granular formulation according to claim 16,
wherein the ratio of L-lysine or L-threonine: riboflavin is 1:
15 0.0001-0.01.
18. The granular formulation according to claim 16 or 17,
wherein the method further comprises removing a bacterial
sludge from the fermented culture mediumof step a), which
20 comprises L-lysine or L-threonine, and riboflavin.
19. A formulation comprising L-lysine or L-threonine, and
riboflavin and prepared by a method comprising:
a) culturing a microorganism which is obtained by
25 modifying a Corynebacterium sp. microorganism having L-lysine
64
or L-threonine-producing ability so as to increase the
production of riboflavin while maintaining the production of Llysine
or L-threonine at a high level, and producing L-lysine
or L-threonine, and riboflavin by a fermentation process; and
5 b) removing a bacterial sludge from the fermented culture
medium of step a), which comprises L-lysine or L-threonine, and
riboflavin.
20. The formulation according to claim 19, wherein the
10 ratio of L-lysine or L-threonine: riboflavin is 1: 0.0001-0.01.
21. A feed additive comprising the granular formulation
of claim 16 or 18 or the formulation of claim 19, which
comprises L-lysine or L-threonine, and riboflavin.