Stress-resistant microorganism and method of producing fermentative product

A microorganism is utilized to fermentatively produce useful substances such as amino acids by cultivating the microorganism in a medium to allow a fermentative product to be produced and accumulated in the medium, and collecting the fermentative product, wherein the microorganism to be used is modified by introduction of at least one of a gene coding for a heat shock protein and a gene coding for a .sigma. factor which specifically functions for the heat shock protein gene to enhance expression amount of the heat shock protein in cells, whereby the microorganism is allowed to have added resistance to stress which would otherwise restrain growth of the microorganism and/or production of the fermentative product.

TECHNICAL FIELD 
The present invention relates to a method of producing a fermentative 
product. In particular, the present invention relates to a method of 
fermentatively producing useful substances such as amino acids by 
utilizing microorganisms, and to microorganisms having added resistance to 
stress which would otherwise restrain growth of the microorganisms and/or 
production of the fermentative products. 
BACKGROUND OF THE INVENTION 
When cells are exposed to stress such as high temperature, high osmotic 
pressure, metabolic inhibition, presence of heavy metal, and viral 
infection, a family of proteins called "heat shock proteins" (hereinafter 
referred to as "HSP") are induced and synthesized in a short period of 
time to cause a defense reaction against the stress. HSP presents broad 
homology ranging from procaryotic cells to eucaryotic cells, and it is 
roughly divided into several groups (HSP 60 group, HSP 70 group, HSP 90 
group, TRiC group, and miscellaneous group) (Hendrick, J. P. and Hartl, F. 
V., Annu. Rev. Biochem., 62, 349-384 (1993)). 
The mechanism of stress resistance exhibited by HSP resides in the function 
of HSP to form higher-order structures of proteins (folding of proteins). 
Namely, when a protein is denatured due to stress, and becomes incapable 
of forming a correct higher-order structure, HSP binds to the protein, and 
the protein is subjected to refolding into the correct higher-order 
structure. Thus the protein can be returned to have its normal function. 
HSP, which functions for the formation of higher-order structures of 
proteins as described above, has been revealed to serve as a molecular 
chaperon not only for denatured proteins but also for cells in a normal 
state through the process of protein folding, assembly, membrane transport 
and so on. Accordingly, its importance is recognized and widely noticed 
(Ellis, R. J. et al., Science, 250, 954-959 (1990)). The term "chaperon" 
means a supporter. This designation results from the fact that HSP binds 
to various proteins, and it exhibits its function. 
Expression of HSP is induced when cells are exposed to stress as described 
above. The induction is usually temporary. It attenuates soon, and a new 
steady state is achieved. It has been revealed that the induction of HSP, 
is made at the transcription level (Cowing, D. C. et al., Proc. Natl. 
Acad. Sci. USA, 80, 2679-2683 (1985); Zhou, Y. N. et al., J. Bacteriol., 
170, 3640-3649 (1988)). It is known that each of the family of HSP genes 
has a promoter structure called "heat shock promoter", and sigma-32 
(.sigma..sup.=) is present which is a .sigma. (sigma) factor to 
specifically function for the heat shock promoter. It is known that 
.sigma..sup.= is a protein encoded by a rpoH gene, having an extremely 
short half-life of about 1 minute, and it closely relates to the temporary 
induction of HSP (Straus, D. B. et al., Nature, 329, 348-351 (1987)). It 
has been revealed that expression control for .sigma..sup.= itself is made 
at the transcription level and at the translation level, however, major 
control is made at the translation level. 
The induction of HSP by heat shock is caused by two mechanisms of increase 
in synthetic amount of .sigma..sup.= and stabilization thereof. Among 
them, as for the increase in synthetic amount of .sigma..sup.=, it has 
been already revealed that the structure of .sigma..sup.= changes due to 
heat, and thus translation is accelerated (Yura, T. et al., Annu. Rev. 
Microbiol., 47, 321-350 (1993)). As for the stabilization of 
.sigma..sup.=, it has been shown that HSP (DnaK or the like) participates 
in degradation of .sigma..sup.=, assuming that feedback control by HSP 
functions (Tilly, K. et al., Cell, 34, 641-646 (1983); Liberek, K., Proc. 
Natl. Acad. Sci. USA, 89, 3516-3520 (1994)). 
As for Escherichia coli (E. coli), it is known that the growth of cells 
relates to HSP in the presence of stress as described above (Meury, J. et 
al., FEMS Microbiol. Lett., 113, 93-100 (1993)). It is also known that 
production of human growth hormone is affected by dnaK, and secretion of 
procollagenase is affected by groE (Hockney, R. C., Trends in 
Biotechnology, 12, 456 (1994)). However, no relationship is known between 
HSP and productivity of fermentative products such as amino acids and 
nucleic acids and the like. As for coryneform bacteria, no relationship is 
known between HSP and growth, and no relationship is also known between 
HSP and productivity of fermentative products. 
DISCLOSURE OF THE INVENTION 
An object of the present invention is to clarify the relationships between 
HSP and growth of microorganisms and between HSP and productivity of 
fermentative products in order to decrease the influence of stress which 
restrains growth of microorganisms and/or production of fermentative 
products so that the productivity and the yield are improved instead of 
being lowered in production of useful substances such as amino acids by 
fermentation. 
As a result of diligent investigations by the present inventors in order to 
achieve the object described above, it has been found out that the 
productivity and the growth can be improved by introducing, into a 
microorganism, a gene coding for HSP or a gene coding for a .sigma. factor 
which specifically functions for the HSP gene, and enhancing expression of 
HSP. Thus the present invention has been completed. 
Namely, the present invention lies in a method of producing a fermentative 
product by utilizing a microorganism, comprising the steps of cultivating 
the microorganism in a medium to allow the fermentative product to be 
produced and accumulated in the medium, and collecting the fermentative 
product, wherein the microorganism is modified by introduction of at least 
one of a gene coding for HSP and a gene coding for a .sigma. factor which 
specifically functions for the HSP gene to enhance expression amount of 
HSP in cells, whereby the microorganism is allowed to have added 
resistance to stress which would otherwise restrain growth of the 
microorganism and/or production of the fermentative product. 
In another aspect, the present invention lies in a microorganism for 
producing a fermentative product, wherein the microorganism is modified by 
introduction of at least one of a gene coding for HSP and a gene coding 
for a .sigma. factor which specifically functions for the HSP gene to 
enhance expression amount of HSP in cells, whereby the microorganism is 
allowed to have added resistance to stress which would otherwise restrain 
growth of the microorganism and/or production of the fermentative product. 
In a preferred embodiment, the method and the microorganism according to 
the present invention deal with various fermentative products including, 
for example, amino acids such as L-threonine, L-lysine, L-glutamic acid, 
L-leucine, L-isoleucine, L-valine, and L-phenylalanine; nucleic acids or 
nucleosides such as guanylic acid, inosine, and inosinic acid; and other 
substances such as vitamins and antibiotics. 
In another preferred embodiment, the stress includes temperature, osmotic 
pressure of the medium, and high concentration of the fermentative product 
which are not preferable for the growth of the microorganism. 
In still another preferred embodiment, the gene coding for the heat shock 
protein specifically includes groE, and the gene coding for the .sigma. 
factor specifically includes rpoH. 
In still another preferred embodiment, the microorganism to which the 
present invention is applied includes bacteria belonging to the genus 
Escherichia, and coryneform bacteria. 
The present invention will be described in detail below. 
The fermentative product to which the present invention is applied is not 
specifically limited provided that it is those produced by fermentation by 
using any microorganism. The fermentative product includes those produced 
by microorganisms including, for example, various L-amino acids such as 
L-threonine, L-lysine, L-glutamic acid, L-leucine, L-isoleucine, L-valine, 
and L-phenylalanine; nucleic acids or nucleosides such as guanylic acid, 
irosine, and inosinic acid; and other substances such as vitamins and 
antibiotics. Even in the case of substances which are not produced at 
present by utilizing microorganisms, the present invention may be applied 
to those substances which will be capable of being produced by 
microorganisms, for example, as a result of success in genetic 
recombination. Among the substances described above, the method of the 
present invention may be preferably applied to those which are secreted to 
the medium to increase the osmotic pressure of the medium, especially such 
as amino acids. 
There is no special limitation to the microorganism which is modified by 
introduction of at least one of the gene coding for HSP and the gene 
coding for the .sigma. factor which specifically functions for the HSP 
gene to enhance expression amount of HSP in cells, thereby being allowed 
to have added resistance to stress that would otherwise restrain growth of 
the microorganism and/or production of the fermentative product, provided 
that the microorganism is those which produce any fermentative product by 
fermentation. The microorganism includes those which have been hitherto 
used for producing substances, including, for example, bacteria belonging 
to the genus Escherichia, coryneform bacteria, bacteria belonging to the 
genus Bacillus, and bacteria belonging to the genus Serratia. A preferable 
microorganism is such one in which a DNA fragment containing a replication 
origin of a plasmid is obtained, the HSP gene or the gene coding for the 
.sigma. factor specific for the HSP gene operates, and the number of 
copies of these genes can be increased in the microorganism. The 
coryneform bacteria described above are a group of microorganisms as 
defined in Bergey's Manual of Determinative Bacteriology, 8th ed., p. 599 
(1974), which are aerobic and non-acid-fast Gram-positive rods having no 
spore-forming ability, including bacteria belonging to the genus 
Corynebacterium, bacteria belonging to the genus Brevibacterium having 
been hitherto classified into the genus Brevibacterium but united at 
present as bacteria belonging to the genus Corynebacterium, and bacteria 
belonging to the genus Brevibacterium closely related to bacteria 
belonging to the genus Corynebacterium. 
Specifically, exemplary microorganisms for each of fermentative products 
are as follows. Those suited for L-threonine include, for example, 
Escherichia coli VKPM B-3996 (RIA 1867) (see U.S. Pat. No. 5,175,107), and 
Corynebactrium acetoacidophilum AJ12318 (FERM BP-1172) (see U.S. Pat. No. 
5,188,949). Those suited for L-lysine include, for example, Escherichia 
coli AJ11442 (NRRL B-12185, FERM BP-1543) (see U.S. Pat. No. 4,346,170), 
Escherichia coli W3110(tyrA) (this strain is obtainable from Escherichia 
coli W3110(tyrA)/pHATerm (FERM BP-3653) by removing a plasmid pHATerm, see 
WO 95/16042 International Publication Pamphlet), Brevibacterium 
lactofermentum AJ12435 (FERM BP-2294) (see U.S. Pat. No. 5,304,476), and 
Brevibacterium lactofermentum AJ3990 (ATCC 31269) (see U.S. Pat. No. 
4,066,501). Those suited for L-glutamic acid include, for example, 
Escherichia coli AJ12624 (FERM BP-3853) (see French Patent Publication No. 
2,680,178), Brevibacterium lactofermentum AJ12821 (FERM BP-4172) (see 
Japanese Patent Laid-open No. 5-26811, and French Patent Publication No. 
2,701,489), Brevibacterium lactofermentum AJ12475 (FERM BP-2922) (see U.S. 
Pat. No. 5,272,067), and Brevibacterium lactofermentum AJ13029 (FERM 
BP-5189) (see JP 95/01586 International Publication Pamphlet). Those 
suited for L-leucine include, for example, Escherichia coli AJ11478 (FERM 
P-5274) (see Japanese Patent Publication No. 62-34397), and Brevibacterium 
lactofermentum AJ3718 (FERM P-2516) (see U.S. Pat. No. 3,970,519). Those 
suited for L-isoleucine include, for example, Escherichia coli KX141 (VKPM 
B-4781) (see European Patent Publication No. 519,113), and Brevibacterium 
flavum AJ12149 (FERM BP-759) (see U.S. Pat. No. 4,656,135). Those suited 
for L-valine include, for example, Escherichia coli VL1970 (VKPM B-4411) 
(see European Patent Publication No. 519,113), and Brevibacterium 
lactofermentum AJ12341 (FERM BP-1763) (see U.S. Pat. No. 5,188,948). Those 
suited for L-phenylanine include, for example, Escherichia coli AJ12604 
(FERM BP-3579) (see Japanese Patent Laid-open No. 5-236947 and European 
Patent Publication No. 488,424), and Brevibacterium lactofermentum AJ12637 
(FERM BP-4160) (see French Patent Publication No. 2,686,898). 
The microorganism to be used for the method of the present invention 
includes the microorganisms as described above, falling under the 
definition that the microorganism is modified by introduction of at least 
one of the gene coding for HSP and the gene coding for the .sigma. factor 
which specifically functions for the HSP gene to enhance expression amount 
of HSP in cells, whereby the microorganism is allowed to have added 
resistance to stress which would otherwise restrain growth of the 
microorganism and/or production of the fermentative product. The gene to 
be introduced into the microorganism may be any one of, or both of the 
gene coding for HSP and the gene coding for the .sigma. factor which 
specifically functions for the HSP gene. 
The phrase "to enhance expression amount of HSP" means the increase in 
amount of HSP production of a microorganism which originally produces HSP, 
and it additionally implies that a microorganism, which does not 
substantially express HSP in its original state, becomes to express HSP. 
Specifically, the enhancement of expression amount of HSP is realized, for 
example, by introducing a foreign or endogenous HSP gene into cells of a 
microorganism, and expressing it therein. In such a procedure, the number 
of copies of the HSP gene in a cell can be increased by using a vector 
autonomously replicable in the microbial cell, especially a plasmid of the 
multiple copy type as the vector. Alternatively, the expression of HSP can 
be also efficiently enhanced by using a promoter having good expression 
efficiency to increase the amount of expression per one unit of the HSP 
gene. Alternatively, HSP can be also enhanced by introducing, into 
microbial cells, a .sigma. factor gene which specifically functions for an 
inherent promoter for the HSP gene. 
The gene coding for HSP includes, for example, groE (gene for GroELS), dnaK 
(gene for DnaK), and dnaJ (gene for DnaJ). Among them, groE is preferred. 
The .sigma. factor, which specifically functions for these genes, is 
exemplified by rpoH which codes for .sigma..sup.=. Microorganisms 
originating these genes are not specifically limited, provided that each 
of the genes is able to function in a cell of microorganism belonging to 
the genus Escherichia or coryneform bacteria, and concretely exemplifed by 
microorganisms belonging to the genus Escherichia and coryneform bacteria. 
The rpoH gene and the groE gene of Escherichia coli have been already 
reported for their nucleotide sequences (rpoH: J. Bacteriol., 170, 
3479-3484 (1988); groE: Nature, 333, 330-334 (1988)). These genes can be 
obtained from Escherichia coli chromosome by means of amplification in 
accordance with a PCR (polymerase chain reaction) method by using 
oligonucleotide primers synthesized on the basis of the sequences. 
Nucleotide sequences of primers for amplifying the rpoh gene are 
exemplified by sequences shown in SEQ ID NOS: 1 and 2. Nucleotide 
sequences of primers for amplifying the groe gene are exemplified by 
sequences shown in SEQ ID NOS: 3 and 4. 
It has been reported that the dnaK gene and the groE gene of Brevibacterium 
flavum are isolated by a PCR method utilizing primers prepared on the 
basis of the amino acid sequence conserved among the dnaK genes or the 
groE genes derived from Escherichia coli and Bacillus subtilis, and these 
genes are highly homologous to the dnaK genes or groE genes derived from 
other microorganisms (Abstracts of Lectures in the 1994 Meeting of the 
Molecular Biology Society of Japan, p. 395). Judging from the fact, it is 
expected that genes coding for the other HSP (dnaJ gene, rpoH gene and the 
like) also have high homology with each of the genes originating from 
Escherichia coli. Therefore, it is thought to be easy to isolate these 
genes from coryneform bacteria by means of hybridization method using the 
genes coding for HSP originating from Escherichia coli, or PCR method 
utilizing a part of the sequence of theses genes. 
In order to introduce the gene obtained as described above into a bacterium 
belonging to the genus Escherichia, for example, a DNA fragment containing 
the gene may be ligated with vector DNA autonomously replicable in cells 
of bacteria belonging to the genus Escherichia, and an obtained 
recombinant vector may be used to transform the bacterium belonging to the 
genus Escherichia. In order to introduce the gene described above into a 
microorganism other than bacteria belonging to the genus Escherichia, a 
DNA fragment containing the gene may be ligated with vector DNA 
autonomously replicable in cells of the microorganism, and an obtained 
recombinant vector may be used to transform the microorganism. 
Plasmid vector DNA is preferred as the vector DNA which can be used in the 
present invention. Those suited for bacteria belonging to the genus 
Escherichia as the microorganism into which the gene is introduced 
include, for example, pUC19, pUC18, pBR322, pHSG299, pHSG399, and RSF1010. 
Alternatively, vectors of phage DNA can be also utilized. In order to 
efficiently achieve expression of HSP, it is available to use promoters 
which operate in microorganisms, such as lac, trp, and PL instead of the 
inherent promoter for the HSP gene. In order to introduce, into the 
microorganism, the HSP gene or the .sigma. factor which specifically 
functions for the HSP gene, DNA containing such a gene may be incorporated 
into chromosome of the microorganism in accordance with a method by using 
transposon (Berg, D. E. and Berg, C. M., Bio/Technol., 1, 417 (1983)), Mu 
phage (Japanese Patent Laid-open No. 2-109985), or homologous 
recombination (Experiments in Molecular Genetics, Cold Spring Harbor Lab. 
(1972)). 
The vector DNA which can be used in the present invention includes plasmid 
vectors autonomously replicable in coryneform bacteria, including, for 
example, pAM 330 (see Japanese Patent Publication No. 1-11280), and 
pHM1519 (see Japanese Patent Laid-open No. 58-77895) when the 
microorganism into which the gene is introduced is a coryneform bacterium. 
The method for transformation is not especially different from ordinary 
ones for preparing transformants of microorganisms. For example, in the 
case of bacteria belonging to the genus Escherichia, transformation can be 
performed in accordance with a method of D. M. Morrison (Methods in 
Enzymology, 68, 326 (1979)), a method for treating recipient cells with 
calcium chloride to increase permeability for DNA (Mandel, M. and Higa, 
A., J. Mol. Biol., 53, 159 (1970)) or the like. Coryneform bacteria can be 
transformed in accordance with the method of Mandel et al. described 
above, or a method for introduction during a proliferating phase (to 
provide so-called competent cells) so that the cells can incorporate DNA 
as reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, 
F. E., Gene, 1, 153 (1977)). Alternatively, recombinant DNA can be also 
introduced after converting DNA recipient cells into protoplasts or 
spheroplasts which easily incorporate recombinant DNA as known for 
Bacillus subtilis, Actinomycetes, and yeast (Chang, S. and Choen, S. N., 
Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, 
O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., 
Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)). Alternatively, recombinant 
DNA can be also introduced into bacteria belonging to the genus 
Brevibacterium or Corynebacterium in accordance with an electric pulse 
method (Sugimoto et al., Japanese Patent Laid-open No. 2-207791). 
Ordinary microorganisms undergo restraint of growth and decrease in 
productivity and yield of fermentative products when they suffer stress 
due to increase in cultivation temperature, high osmotic pressure caused 
by fermentative products or high concentration medium components, or 
metabolic abnormality associated with production of aimed fermentative 
products. On the contrary, it is possible to add resistance to such stress 
by enhancing expression of HSP. As a result, it is possible to increase 
the productivity of fermentative products in environments in which 
microorganisms suffer stress as described above. The resistance to stress 
does not mean complete resistance, and hence it also includes properties 
to decrease influences caused by the stress. Both of the restraint of 
growth and the decrease in productivity and yield of fermentative products 
are not necessarily desensitized depending on the type of genes to be 
introduced and the type of host microorganisms. There is sometimes a case 
in which the yield of a fermentative product is improved although the 
growth is restrained. The stress, against which the resistance can be 
added in accordance with the method of the present invention, includes, 
for example, temperature, osmotic pressure of a medium, and high 
concentration amino acid in a medium which are not preferable for the 
growth of the microorganism. 
The medium for production by fermentation to be used for the method of the 
present invention may be well-known media having been hitherto used 
depending on microorganisms to be utilized. Namely, it is an ordinary 
medium containing a carbon source, a nitrogen source, inorganic ions, and 
optionally other organic components. No special medium is required to 
carry out the present invention. 
Those which are usable as the carbon source include, for example, sugars 
such as glucose, lactose, galactose, fructose, and starch hydrolysate; 
alcohols such as glycerol and sorbitol; and organic acids such as fumaric 
acid, citric acid, and succinic acid. 
Those which are usable as the nitrogen source include, for example, 
inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and 
ammonium phosphate; organic nitrogen such as soybean hydrolysate; ammonia 
gas; and aqueous ammonia. 
It is desirable to contain, as organic trace nutrient sources, required 
substances such as vitamin B.sub.1, L-homoserine, and L-tyrosine; yeast 
extract or the like in appropriate amounts. Besides them, potassium 
phosphate, magnesium sulfate, iron ion, manganese ion and so on are added 
in small amounts, if necessary. 
Cultivation may be performed under well-known conditions having been 
hitherto used depending on microorganisms to be utilized. Cultivation is 
preferably performed, for example, under an aerobic condition for 16 to 
120 hours. The cultivation temperature is controlled at 25.degree. C. to 
45.degree. C., and pH is controlled at 5 to 8 during the cultivation. For 
pH adjustment, it is possible to use inorganic or organic, acidic or 
alkaline substances, ammonia gas and so on. 
In the present invention, the metabolic product is collected from a medium 
liquid after completion of the cultivation with no necessity for any 
special method. Namely, the present invention can be carried out by 
combining methods of ion exchange resin, precipitation and others having 
been hitherto well-known.

BEST MODE FOR CARRYING OUT THE INVENTION 
The present invention will be described in more detail below with reference 
to Examples. 
EXAMPLE 1 
Preparation of Plasmids for Introducing rpoH Gene and groE Gene 
&lt;1&gt; Cloning of rpoH Gene and groe Gene 
A rpoH gene and a groE gene of Escherichia coli were cloned in accordance 
with the PCR (polymerase chain reaction) method. Primers used in the PCR 
method were synthesized on the basis of sequences of the rpoh gene (J. 
Bacteriol., 170, 3479-3484 (1988)) and the groE gene (Nature, 333, 330-334 
(1988)) of Escherichia coli already reported. Oligonucleotides having 
nucleotide sequences shown in SEQ ID NO: 1 (5' side) and SEQ ID NO: 2 (3' 
side) were synthesized as primers for amplifying the rpoH gene. 
Oligonucleotides having nucleotide sequences shown in SEQ ID NO: 3 (5' 
side) and SEQ ID NO: 4 (3' side) were synthesized as primers for 
amplifying the groE gene. 
Primers for amplifying rpoH gene 
5' side: 5'-CGGAACGAAGTTTGATATCA-3' (SEQ ID NO: 1) 
3' side: 5'-ATCCAGGGTTCTCTGCTTAA-3' (SEQ ID NO: 2) 
Primers for amplifying groE gene 
5' side: 5'-GACGTCGATAGCAGGCCAAT-3' (SEQ ID NO: 3) 
3' side: 5'-GACGCACTCGCGTCGTCCGT-3' (SEQ ID NO: 4) 
Chromosomal DNA was extracted from E. coli K-12 strain in accordance with a 
method of Saito et al. (Saito, H. and Miura, K., Biochem. Biophys. Acta., 
72, 619 (1963)), and it was used as a template to perform PCR by using the 
oligonucleotides described above as the primers. 
The reaction was repeated over 25 cycles in PCR, each cycle comprising heat 
denaturation (94.degree. C., 1 minute), annealing (37.degree. C., 2 
minutes), and polymerase reaction (72.degree. C., 3 minutes). 
Both ends of obtained amplified products were blunt-ended by using a 
commercially available DNA blunt end formation kit (produced by Takara 
Shuzo, Blunting kit). After that, the products were respectively cloned 
into a HincII site of a vector plasmid pSTV28 (produced by Takara Shuzo) 
to obtain plasmids pSTV28-rpoH and pSTV28-groE. 
&lt;2&gt; Introduction of Replication Origin From Coryneform Bacterium into 
Plasmid Containing rpoH gene and Plasmid Containing groE Gene 
In order to make pSTV28-rpoH to be autonomously replicable in cells of 
coryneform bacteria, a replication origin (Japanese Patent Laid-open No. 
5-007491) originating from an already obtained plasmid pHM1519 
autonomously replicable in coryneform bacteria (Miwa, K. et al., Agric. 
Biol. Chem., 48 (1984) 2901-2903) was introduced into pSTV28-rpoH. 
Specifically, pHM1519 was digested with restriction enzymes BamHI and KpnI 
to obtain a DNA fragment containing the replication origin. The obtained 
fragment was blunt-ended by using a DNA blunt end formation kit (produced 
by Takara Shuzo, Blunting kit), and then a KpnI linker (produced by Takara 
Shuzo) was ligated with its ends. The fragment was inserted into a KpnI 
site of pSTV28-rpoh to obtain pRPH. On the other hand, a plasmid pHSG399 
having no rpoH gene was used as a control. The replication origin 
originating from pHM1519 was also inserted into a SalI site of the control 
plasmid by using a SalI linker (produced by Takara Shuzo) to obtain pSAC4. 
EXAMPLE 2 
Production of L-Glutamic Acid by L-Glutamic Acid-producing Bacterium with 
Introduced rpoH Gene 
pRPH and pSAC4 prepared as described above were introduced into 
Brevibacterium lactofermentum AJ12821 (FERM BP-4172) having L-glutamic 
acid-producing ability. L-Glutamic acid productivity of transformants 
containing each of the introduced plasmids was evaluated. The plasmids 
were introduced into cells of Brevibacterium lactofermentum by using an 
electric pulse method (Japanese Patent Laid-open No. 2-207791). 
The transformants containing the introduced plasmids were selected on a 
CM2G plate medium (containing 10 g of polypeptone, 10 g of yeast extract, 
5 g of glucose, 5 g of NaCl, and 15 g of agar in 1 L of pure water, pH 
7.2) containing 4 .mu.g/ml of chloramphenicol. 
The L-glutamic acid productivity of the obtained transformants was 
evaluated as follows. Each of the transformants was cultivated on the CM2G 
plate medium to refresh cells. Each of the refreshed transformants was 
cultivated at 35.degree. C. for 20 hours in a medium containing 80 g of 
glucose, 1 g of KH.sub.2 PO.sub.4, 0.4 g of MgSO.sub.4.7H.sub.2 O, 30 g of 
(NH.sub.4).sub.2 SO.sub.4, 0.01 g of FeSO.sub.4.7H.sub.2 O, 0.01 g of 
MnSO.sub.4.7H.sub.2 O, 15 ml of soybean hydrolysate solution, 200 .mu.g of 
thiamine hydrochloride, 300 .mu.g of biotin, 4 mg of chloramphenicol, and 
50 g of CaCO.sub.3 in 1 L of pure water (at pH adjusted to 8.0 with KOH). 
Usually, Brevibacterium lactofermentum is preferably cultivated at a 
cultivation temperature of 31 to 32.degree. C. 
The bacterial cell concentration, and the amount of L-glutamic acid 
accumulated in the medium were measured after the cultivation. L-Glutamic 
acid was quantitatively determined by using Biotec Analyzer AS-210 
produced by Asahi Chemical Industry. The bacterial cell concentration was 
determined by measuring the absorbance at 660 nm (OD660) of the culture 
liquid diluted 51 times with 0.2 N hydrochloric acid. Results are shown in 
Table 1. 
TABLE 1 
______________________________________ 
L-Glutamic acid 
Bacterial strain (g/dl) OD 
.sub.660 
______________________________________ 
AJ12821/pSAC4 3.3 0.98 
AJ12821/pRPH 4.3 0.68 
______________________________________ 
As clarified from the results, although the growth was restrained, the 
L-glutamic acid productivity was improved in the L-glutamic acid-producing 
bacterium of Brevibacterium lactofermentum containing the introduced rpoH 
gene. 
EXAMPLE 3 
Production of L-Lysine by L-Lysine-producing Bacterium with Introduced rpoH 
Gene 
pRPH and pSAC4 prepared as described above were introduced into 
Brevibacterium lactofermentum AJ12435 (FERM BP-2294) exhibiting resistance 
to S-(2-aminoethyl)-L-cysteine and having L-lysine-producing ability 
derived by mutation from Brevibacterium lactofermentum ATCC 13869. 
L-Lysine productivity of transformants harboring each of the introduced 
plasmids was evaluated. 
The plasmids were introduced into cells of Brevibacterium lactofermentum by 
using an electric pulse method (Japanese Patent Laid-open No. 2-207791). 
The transformants harboring the plasmids were selected on a CM2G plate 
medium (containing 10 g of polypeptone, 10 g of yeast extract, 5 g of 
glucose, 5 g of NaCl, and 15 g of agar in 1 L of pure water, pH 7.2) 
containing 4 .mu.g/ml of chloramphenicol. 
The L-lysine productivity of the obtained transformants was evaluated as 
follows. Each of the transformants was cultivated on the CM2G plate medium 
to refresh cells. Each of the refreshed transformants was cultivated at 
31.5.degree. C. for 60 hours in a medium containing 100 g of glucose, 1 g 
of KH.sub.2 PO.sub.4, 0.4 g of MgSO.sub.4.7H.sub.2 O, 30 g of 
(NH.sub.4).sub.2 SO.sub.4, 0.01 g of FeSO.sub.4.7H.sub.2 O, 0.01 g of 
MnSO.sub.4.7H.sub.2 O, 15 ml of soybean hydrolysate solution, 200 .mu.g of 
thiamine hydrochloride, 300 .mu.g of biotin, 4 mg of chloramphenicol, and 
50 g of CaCO.sub.3 in 1 L of pure water (at pH adjusted to 8.0 with KOH). 
The bacterial cell concentration, and the amount of L-lysine accumulated 
in the medium were measured after the cultivation. L-Lysine was 
quantitatively determined as L-lysine hydrochloride by using Biotec 
Analyzer AS-210 produced by Asahi Chemical Industry. The bacterial cell 
concentration was determined by measuring the absorbance at 660 nm 
(OD.sub.660) of the culture liquid diluted 51 times with 0.2 N 
hydrochloric acid. Results are shown in Table 2. 
TABLE 2 
______________________________________ 
L-Lysine hydrochloride 
Bacterial strain (g/L) OD.sub.660 
______________________________________ 
AJ11446/pSAC4 22 0.78 
AJ11446/pRPH 27 1.15 
______________________________________ 
As shown in Table 2, the growth was good, and the L-lysine productivity was 
also improved in the L-lysine-producing bacterium of Brevibacterium 
lactofermentum containing the introduced rngH gene. It is supposed that 
this result arose from mitigation of the influence of increased osmotic 
pressure due to accumulation of L-lysine. 
EXAMPLE 4 
Production of L-Phenylalanine by L-Phenylalanine-producing Escherichia coli 
with Introduced groE Gene 
pSTV28-groE or pSTV28 prepared as described above was introduced into a 
phenylalanine-producing bacterium, AJ12604 strain (FERM BP-3579) bred from 
Escherichia coli K-12 strain. L-Phenylalanine productivity of 
transformants harboring each of the introduced plasmids was evaluated. 
The plasmids were introduced by using an electric pulse method (Japanese 
Patent Laid-open No. 2-207791). The transformants harboring the plasmids 
were selected on an L plate medium (containing 10 g of polypeptone, 5 g of 
yeast extract, 5 g of NaCl, and 15 g of agar in 1 L of pure water, pH 7.2) 
containing 40 .mu.g/ml of chloramphenicol. 
The L-phenylalanine productivity of the obtained transformants was 
evaluated as follows. The transformants were cultivated on the L plate 
medium containing 40 mg/L of chloramphenicol to refresh cells. Each of the 
refreshed transformants was cultivated at 45.degree. C. for 40 hours in a 
medium containing 20 g of glucose, 29.4 g of Na.sub.2 HPO.sub.4, 6 g of 
KH.sub.2 PO.sub.4, 1 g of NaCl, 2 g of NH.sub.4 Cl, 10 g of sodium 
citrate, 0.4 g of sodium glutamate, 3 g of MgSO.sub.4.7H.sub.2 O, 0.23 g 
of KCl, 2 mg of thiamine hydrochloride, 75 mg of L-tyrosine, and 40 mg of 
chloramphenicol in 1 L of pure water (at pH adjusted to 7.0 with KOH). The 
bacterial cell concentration, and the amount of L-phenylalanine 
accumulated in the medium were measured after the cultivation. 
L-Phenylalanine was quantitatively determined by using Biotec Analyzer 
AS-210 produced by Asahi Chemical Industry. The bacterial cell 
concentration was determined by measuring the absorbance at 660 nm 
(OD.sub.660) of the culture liquid diluted 51 times with pure water. 
Results are shown in Table 3. 
TABLE 3 
______________________________________ 
L-Phenylalanine 
Bacterial strain (g/L) OD.sub.660 
______________________________________ 
AJ12604/pSTV28 1.5 0.143 
AJ12604/PSTV2B-groE 2.0 0.146 
______________________________________ 
According to the results, it is clear that the productivity of 
L-phenylalanine was improved in E. coli with the introduced groE gene. 
EXAMPLE 5 
Production of L-Lysine by L-Lysine-producing Escherichia coli with 
Introduced rpoh Gene 
Escherichia coli W3110 (tyrA) strain was used as a host for L-lysine 
production. W3110 (tyrA) strain is described in detail in European Patent 
Publication No. 488424 (1992). However, its preparation method will be 
briefly described as follows. 
E. coli W3110 strain was obtained from National Institute of Genetics 
(Mishima-shi, Shizuoka-ken, Japan). This strain was spread over an LB 
plate containing streptomycin, and strains which formed colonies were 
selected to obtain a streptomycin-resistant strain. The selected 
streptomycin-resistant strain was mixed with E. coli K-12 ME8424 strain to 
induce conjugation by stationarily cultivating them for 15 minutes under 
conditions at 37.degree. C. in a complete medium (L-Broth: 1% Bacto 
trypton, 0.5% Yeast extract, 0.5% NaCl). E. coli K-12 ME8424 strain has 
inherited characters of (HfrP045, thi, relAl, tyrA::Tn10, ung-1, nadB), 
and it is obtainable from National Institute of Genetics. After the 
conjugation, the culture was spread over a complete medium (L-Broth: 1% 
Bacto trypton, 0.5% Yeast extract, 0.5% NaCl, 1.5% agar) containing 
streptomycin, tetracycline, and L-tyrosine to select a strain which formed 
colonies. This strain was designated as E. coli W3110 (tyrA). 
Many strains, which were formed by introducing plasmids into this strain, 
are described in European Patent Publication No. 488424 (1992). For 
example, a strain, which was obtained by introducing a plasmid pHATerm, 
was designated as Escherichia coli W3110 (tyrA)/pHATerm, was deposited in 
National Institute of Bioscience and Human Technology of Agency of 
Industrial Science and Technology (1-3, Higashi 1-chome, tsukuba-shi, 
ibaraki-ken, 305 Japan) based on Budapest Treaty on Nov. 16, 1991, as 
accession number of FERM BP-3653. Escherichia coli W3110 (tyrA) strain can 
be obtained by removing the plasmid pHATerm from this bacterial strain by 
using an ordinary method. 
A plasmid RSFD80 having a gene for lysine biosynthesis was introduced into 
the Escherichia coli W3110 (tyrA) strain in accordance with the method 
described in Example 4. RSFD80 is described in WO 95/16042 International 
Publication Pamphlet, and it contains DNA coding for dihydrodipicolinate 
synthase with desensitized feedback inhibition by L-lysine, and DNA coding 
for aspartokinase with desensitized feedback inhibition by L-lysine. E. 
coli JM109 strain harboring the RSFD80 plasmid was designated as AJ12396, 
and was deposited in National Institute of Bioscience and Human Technology 
of Agency of Industrial Science and Technology (1-3, Higashi 1-chome, 
tsukuba-shi, ibaraki-ken, ken, 305 Japan) on Oct. 28, 1993, as accession 
number of FERM P-13936, and transferred from the original deposition to 
international deposition based on Budapest Treaty on Nov. 1, 1994, and has 
been deposited as accession number of FERM BP-4859. 
A transformant of the W3110 (tyrA) strain, in which PSFD80 was introduced, 
was selected on a plate medium containing 50 .mu.g/ml of streptomycin. 
On the other hand, a plasmid for introducing the rpoH gene was constructed 
as follows. The rpoH gene was amplified by the PCR method in accordance 
with the method described in the item &lt;1&gt; in Example 1. An obtained 
amplified product was blunt-ended at its both ends by using a commercially 
available DNA blunt end formation kit (produced by Takara Shuzo, Blunting 
kit), and then it was cloned into a HincII site of a vector plasmid pMW119 
(produced by Wako Pure Chemical Industries) to obtain a plasmid pMWrpoH. 
This plasmid was introduced into Escherichia coli W3110 (tyrA)/RSFDB80 
strain in accordance with the method described above. A transformant 
containing the introduced plasmid was selected on an L plate medium 
containing 50 .mu.g/ml of streptomycin and 50 .mu.g/ml of ampicillin. 
The L-lysine productivity was evaluated for Escherichia coli W3110 
(tyrA)/RSFD80 strain, and Escherichia coli W3110 (tyrA)/RSFD80+pMWrpoH 
strain obtained as described above. 
The L-lysine productivity of the obtained transformants was evaluated as 
follows. The transformants were cultivated on the L plate medium to 
refresh cells. Each of the refreshed transformants was cultivated at 
37.degree. C. for 30 hours in a medium containing 40 g of glucose, 1 g of 
KH.sub.2 PO.sub.4, 0.01 g of MnSO.sub.4.7H.sub.2 O, 0.01 g of 
FeSO.sub.4.7H.sub.2 O, 2 g of yeast extract, 0.1 g of L-tyrosine, 1 g of 
MgSO.sub.4.7H.sub.2 O, and 25 g of CaCO.sub.3 in 1 L of pure water (at pH 
adjusted to 7.0 with KOH). In this experiment, the transformants were also 
cultivated in the same medium except that 40 g/L of L-lysine hydrochloride 
was initially added. L-Lysine was quantitatively determined by using 
Biotec Analyzer AS-210 produced by Asahi Chemical Industry. Table 4 shows 
amounts of increase in L-lysine (obtained by subtracting the amount of 
initially added L-lysine from the amount of L-lysine in the medium after 
the cultivation) as amounts of L-lysine hydrochloride. 
TABLE 4 
______________________________________ 
Production amount of L-lysine 
hydrochloride (g/L) 
Initially added L-lysine hydrochloride 
Bacterial (g/L) 
strain 0 40 
______________________________________ 
W3110(tyrA)/RSFD80 
9.17 6.43 
W3110(tyrA)/RSFD80 9.22 7.64 
______________________________________ 
+pMWrpoH 
According to the results, it is clear that the L-lysine productivity was 
improved in Escherichia coli containing the introduced rpoH gene even in 
the presence of the high concentration of L-lysine as compared with the 
strain containing no introduced rpoH gene. 
Industrial Applicability 
The relationships between HSP and growth of microorganisms and between HSP 
and productivity of fermentative products have been clarified by the 
present invention. Thus it is possible to decrease the influence of stress 
and improve deterioration of productivity and yield in fermentative 
production of useful substances such as amino acids. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - &lt;160&gt; NUMBER OF SEQ ID NOS: 4 
- - &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 20 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
synthetic DNA 
- - &lt;400&gt; SEQUENCE: 1 
- - cggaacgaag tttgatatca - # - # 
- # 20 
- - - - &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 20 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
synthetic DNA 
- - &lt;400&gt; SEQUENCE: 2 
- - atccagggtt ctctgcttaa - # - # 
- # 20 
- - - - &lt;210&gt; SEQ ID NO 3 
&lt;211&gt; LENGTH: 20 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
synthetic DNA 
- - &lt;400&gt; SEQUENCE: 3 
- - gacgtcgata gcaggccaat - # - # 
- # 20 
- - - - &lt;210&gt; SEQ ID NO 4 
&lt;211&gt; LENGTH: 20 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
synthetic DNA 
- - &lt;400&gt; SEQUENCE: 4 
- - gacgcactcg cgtcgtccgt - # - # 
- # 20 
__________________________________________________________________________