DNA base sequence, method for preparing a recombinant plasmid including the DNA base sequence, and breeding method for enhancing the protein-secreting ability of a microorganism by introducing thereinto the recombinant plasmid

This invention relates to a DNA base sequence, capable of increasing the amount of protein secreted by microorganisms, and its derivative sequences; a recombinant plasmid including the whole or a part of said DNA base sequence; a method for preparing the recombinant plasmid in which, when microorganisms having introduced thereinto a recombinant DNA including the desired DNA base sequence are to be separated in the process of cloning, suitable transformants can be efficiently selected by taking the amount of protein secreted out of the cells and particularly the activity of an enzyme protein as an index; and a method of microbial breeding which comprises introducing the recombinant plasmid into a microorganism to increase the amount of protein secreted by the microorganism.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a DNA base sequence capable of increasing the 
amount of protein secreted by microorganisms and its derivative sequences; 
a recombinant plasmid including the whole or a part of said DNA base 
sequence; a method for preparing the recombinant plasmid; and a method of 
microbial breeding which comprises introducing the recombinant plasmid 
into a microorganism to increase the amount of protein secreted by the 
microorganism. 
2. Description of the Prior Art 
Some microorganisms (e.g., bacteria, yeasts and the like) secrete protein 
into the periplasm or out of the cells. Among others, bacteria of the 
genus Bacillus secrete a large amount of protein out of the cells. 
From the viewpoint of microbial production of protein, it is of great 
significance to incorporate the genetic information involved in the 
promotion of protein secretion by such microorganisms into a host vector 
system according to genetic engineering techniques and thereby create a 
host vector system having the genetic information involved in the 
promotion of protein secretion. That is, the gene associated with high 
amounts of protein secretions can be expressed by forming such a host 
vector system within a microorganism. If the microorganism having amounts 
of protein secretions ability imparted thereto is grown, for example, in a 
culture medium, a large amount of protein is secreted into the culture 
medium. This protein can then be recovered from the culture medium 
according to a simple procedure. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a DNA base sequence, 
capable of increasing the amount of protein secreted by microorganisms, 
and its derivative sequences. 
It is another object of the present invention to provide a recombinant 
plasmid including the whole or a part of said base sequence and an 
efficient method for preparing this recombinant plasmid. 
It is still another object of the present invention to provide a method of 
microbial breeding for increasing the amount of protein secreted by a 
microorganism by introducing thereinto such a recombinant plasmid. 
These objects of the present invention are accomplished by 
(a) a DNA base sequence capable of increasing the amount of protein 
secreted in which one of the strand comprises bases arranged in the 
following order: 
______________________________________ 
A T C G A T A C G C T T C T C C A G A G 
A G A G C T T G A G G A C A T A G T C A 
A A G A A C T G C C T T T A A T T G A T 
G A A G T C G G A C A G G C G C T G C T 
C G G T T A T G A A A A C G A T T A T T 
A C A T G A T G C T C G G G C T C G T C 
A A A G C C A T T G A A T G C A A C A A 
C T G G G A C T G T G A C G A G T G G G 
G A A A A G A A C T G G A C A A A G A A 
G A A G C A T A T G A A T G T T A C T T 
A C A G G C G A T C G A A T G G T G C C 
G G C A G C T T A T C G T G A A T T G A 
G G C G C T T T A A G C C G A C C A C C 
T G A G G G C G A T G A T G A A C C G G 
C T T T G C T C T C T G A A C A G A C T 
G A C A C G G A T G C C G T C G G G A T 
T A A A T G T C A C A T C A A A A T C A 
T T T T T G A T G C C G G T C T G C T T 
A A T G A G C C G C T T G A C C G C C G 
C G G C G T C A G A T G A T T G C G C T 
G C G G T G A G G A T G C G G C G C G C 
A A G C T C T T T T G A C G C C G A C A 
G C C G C G C A A G G A G C A G A C G G 
G C G T C C T C C A G C A T G C C C G C 
A G C C T C T T T T G C G G A C G C T G 
T C A A A A T A T C T G C G T G A A T C 
G T G G G A G C A G G C G C A G G A T T 
G C G T A T G G C G C G G C G A A A A C 
A G G A C A G C A T G G A T A C A C A T 
A A A G C A T G C G G C G G C C T C C T 
T C T G A G A T A A C A T T C G T T A T 
A C A T G A G T A T A G G C G G C G T G 
A T A A C G A G T T A T G A C A T G C A 
A A A A G A C C A C A A T G C G G G T G 
T T G C G G T C T T T T C G G T G T T T 
G T C G G T G G T T A T G C G A C G C T 
G T T C G C C C A T T C T C T T T T G A 
A A T T G C G A C G T C A G G G A C T A 
T A G T C C T T A G C G G T T T G T C G 
G A A A A C C G T T A A A A A A C C A G 
C A G A A C C A C C A G A T T G A T C T 
G C T T C A T C C C A A A C G T C T G C 
C T T T A T G G T A G T T A T A T A G T 
C C T G T T C G C C A A A T G C T C T G 
T T C G G G A C T A T G G G A T T A C C 
G T G G T T T G C G G T G T C A C G C A 
G A T A C T T T T A C A C A T A C T T T 
T C G G T G A A A A A T C C C G C A A A 
A A C G T T T A C A C T A T T A G T A A 
C A G A T C A A A T A C C T A G G A C T 
C G T T C A C C A T A C A C A A T T C A 
T T G A T C T T T C A A A A A A A G G A 
G T G T G G A A A C G G T G G A A A A G 
A A A T T A G A A G A A G T A A A G C A 
A T T A T T A T T C C G A C T T G A A A 
A T G A T A T C A G A G A A A C A A C C 
G A C T C A T T A C G A A A C A T T A A 
C A A A A G C A T T G A T C A G C T C G 
A T A A A T T C T C A T A T G C A A T G 
A A A A T T T C T T A A A A A G A C T T 
G G A A A C A A G T C T T T T T T T T G 
T G C A T T T T T C A C C C A T T T C A 
T G G A T A A A G T A T T A T A C G A T 
T G T T A A A A A A C G A A A A A C C T 
G C T G T C T T T C A T C A C C T G C A 
T T T A G T A A A A T A G A A T G G G A 
G G G T G A A G A C A A T T A T T G A G 
C A A A T G T G T T T A G A T G C C G A 
A A C G A T T A A A G G G A A G A T G A 
A G G A A A T T G T T G G G G A T A A A 
G T C G A T A A T C T A C A T T T A G A 
A G A G A C T C T T T T G A C C T T C A 
T T A A T G A A A A G A A G C A C T T T 
T C A T T C G G T G T C C T T G C T T T 
C C A G C A T T A T G T T G C T T T T A 
A G G G T A C A C A T T C C T C G G A A 
A T C A C A C T A C T G G C C G C T G G 
A A T T G A A C T T T T A A T T T T A G 
C T T T T G A T A T T T T T G A C G A T 
A T T G A A G A T G A A G A T A A C T T 
T A A T A A G G C A T G G A T G C A A A 
C T G A C C A T G C T A T A T C C C T G 
A A T G C G G C T A C T T C T C T G T A 
T T C A A T A A G C C T G C A A G C C A 
T T T G T G A G C T T G A A T C A A A C 
A A T C G A T 
______________________________________ 
where A, T, C and G represent adenine, thymine, cytosine and guanine, 
respectively, and derivative sequences thereof; 
(b) a recombinant plasmid including the whole or a part of the aforesaid 
DNA base sequence or a derivative sequence thereof; 
(c) a method for preparing the aforesaid recombinant plasmid in which, when 
microorganisms having introduced thereinto a recombinant DNA including the 
desired DNA base sequence are to be separated in the process of cloning, 
suitable transformants can be very efficiently selected by paying 
attention to an enzyme protein secreted out of the cells and determining 
the amount thereof and particularly the extracellular enzyme activity 
thereof to find transformants having markedly increased amounts of protein 
secretion; and (d) a method of microbial breeding which comprises 
introducing the aforesaid recombinant plasmid into a microorganism to 
increase the amount of protein secreted by the microorganism. 
The phrase "enhancement of the protein-secreting ability of a 
microorganism" means an increasing of the level of a secreted protein from 
a microorganism and the phrase "separate coexistence" means of the DNA 
sequence and a gene encoding the secreted protein in the bacterium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In accordance with the present invention, the DNA base sequence capable of 
increase the amount of protein secreted by microorganisms is one which 
defines, from the 5' to the 3' terminus, the following arrangement of 
bases: 
______________________________________ 
A T C G A T A C G C T T C T C C A G A G 
A G A G C T T G A G G A C A T A G T C A 
A A G A A C T G C C T T T A A T T G A T 
G A A G T C G G A C A G G C G C T G C T 
C G G T T A T G A A A A C G A T T A T T 
A C A T G A T G C T C G G G C T C G T C 
A A A G C C A T T G A A T G C A A C A A 
C T G G G A C T G T G A C G A G T G G G 
G A A A A G A A C T G G A C A A A G A A 
G A A G C A T A T G A A T G T T A C T T 
A C A G G C G A T C G A A T G G T G C C 
G G C A G C T T A T C G T G A A T T G A 
G G C G C T T T A A G C C G A C C A C C 
T G A G G G C G A T G A T G A A C C G G 
C T T T G C T C T C T G A A C A G A C T 
G A C A C G G A T G C C G T C G G G A T 
T A A A T G T C A C A T C A A A A T C A 
T T T T T G A T G C C G G T C T G C T T 
A A T G A G C C G C T T G A C C G C C G 
C G G C G T C A G A T G A T T G C G C T 
G C G G T G A G G A T G C G G C G C G C 
A A G C T C T T T T G A C G C C G A C A 
G C C G C G C A A G G A G C A G A C G G 
G C G T C C T C C A G C A T G C C C G C 
A G C C T C T T T T G C G G A C G C T G 
T C A A A A T A T C T G C G T G A A T C 
G T G G G A G C A G G C G C A G G A T T 
G C G T A T G G C G C G G C G A A A A C 
A G G A C A G C A T G G A T A C A C A T 
A A A G C A T G C G G C G G C C T C C T 
T C T G A G A T A A C A T T C G T T A T 
A C A T G A G T A T A G G C G G C G T G 
A T A A C G A G T T A T G A C A T G C A 
A A A A G A C C A C A A T G C G G G T G 
T T G C G G T C T T T T C G G T G T T T 
G T C G G T G G T T A T G C G A C G C T 
G T T C G C C C A T T C T C T T T T G A 
A A T T G C G A C G T C A G G G A C T A 
T A G T C C T T A G C G G T T T G T C G 
G A A A A C C G T T A A A A A A C C A G 
C A G A A C C A C C A G A T T G A T C T 
G C T T C A T C C C A A A C G T C T G C 
C T T T A T G G T A G T T A T A T A G T 
C C T G T T C G C C A A A T G C T C T G 
T T C G G G A C T A T G G G A T T A C C 
G T G G T T T G C G G T G T C A C G C A 
G A T A C T T T T A C A C A T A C T T T 
T C G G T G A A A A A T C C C G C A A A 
A A C G T T T A C A C T A T T A G T A A 
C A G A T C A A A T A C C T A G G A C T 
C G T T C A C C A T A C A C A A T T C A 
T T G A T C T T T C A A A A A A A G G A 
G T G T G G A A A C G G T G G A A A A G 
A A A T T A G A A G A A G T A A A G C A 
A T T A T T A T T C C G A C T T G A A A 
A T G A T A T C A G A G A A A C A A C C 
G A C T C A T T A C G A A A C A T T A A 
C A A A A G C A T T G A T C A G C T C G 
A T A A A T T C T C A T A T G C A A T G 
A A A A T T T C T T A A A A A G A C T T 
G G A A A C A A G T C T T T T T T T T G 
T G C A T T T T T C A C C C A T T T C A 
T G G A T A A A G T A T T A T A C G A T 
T G T T A A A A A A C G A A A A A C C T 
G C T G T C T T T C A T C A C C T G C A 
T T T A G T A A A A T A G A A T G G G A 
G G G T G A A G A C A A T T A T T G A G 
C A A A T G T G T T T A G A T G C C G A 
A A C G A T T A A A G G G A A G A T G A 
A G G A A A T T G T T G G G G A T A A A 
G T C G A T A A T C T A C A T T T A G A 
A G A G A C T C T T T T G A C C T T C A 
T T A A T G A A A A G A A G C A C T T T 
T C A T T C G G T G T C C T T G C T T T 
C C A G C A T T A T G T T G C T T T T A 
A G G G T A C A C A T T C C T C G G A A 
A T C A C A C T A C T G G C C G C T G G 
A A T T G A A C T T T T A A T T T T A G 
C T T T T G A T A T T T T T G A C G A T 
A T T G A A G A T G A A G A T A A C T T 
T A A T A A G G C A T G G A T G C A A A 
C T G A C C A T G C T A T A T C C C T G 
A A T G C G G C T A C T T C T C T G T A 
T T C A A T A A G C C T G C A A G C C A 
T T T G T G A G C T T G A A T C A A A C 
A A T C G A T 
______________________________________ 
where A, T, C and G represent adenine, thymine, cytosine and guanine, 
respectively. 
The term "derivative sequences of the DNA base sequence" as used herein 
means derivative sequences of the aforesaid DNA base sequence in which the 
DNA base sequence has undergone base substitution, deletion, transposition 
and/or insertion without impairing its capability of increasing the amount 
of protein secreted by microorganisms. 
The aforesaid DNA base sequence capable of increasing the amount of protein 
secreted by microorganisms (hereinafter referred to as "the DNA base 
sequence of the present invention") can be obtained by cloning from the 
chromosomal DNA of a microorganism having the ability to secrete protein 
or by chemical synthesis. 
Preferred chromosomal DNA sources for obtaining the DNA base sequence of 
the present invention include chromosomal DNA possessed by Bacillus 
bacteria having the ability to secrete various types of proteins in 
considerable amounts, such as Bacillus amyloliquefaciens ATCC 23842* or 
ATCC 23350*; Bacillus subtilis ATCC e6051*; or Bacillus licheniformis ATCC 
21415*. 
FNT *A stock strain maintained in the American Type Culture Collection, 12301 
Parklawn Drive Rockville, MD 10852-1776, U.S.A. 
By way of example, the procedure for obtaining the DNA base sequence of the 
present invention from a Bacillus bacterium will be described hereinbelow. 
First, DNA is isolated from cells of a Bacillus bacterium according to a 
conventional procedure. This DNA is fragmented either by means of a 
restriction enzyme or by a physical shearing action. It is to be 
understood that, if a restriction enzyme is used, it should be selected so 
as not to cleave the base sequence involved in the promotion of protein 
secretion. Even though the base sequence is inevitably cleaved by the 
restriction enzyme, care must be taken that the base sequence undergoes 
only partial degradation. 
Then, the resulting DNA fragments are linked with plasmid DNA or phage DNA 
used as the vector. One example of an enzyme useful for this purpose is 
T.sub.4 DNA ligase. Depending on the form of the DNA fragments to be 
linked, it may be desirable to use a linker according to the need. The 
hybrid DNA thus obtained may be introduced into a Bacillus bacterium 
according to any of the commonly used methods. By way of example, the 
hybrid DNA can be efficiently introduced into cells of a Bacillus 
bacterium by using the protoplast transformation method or the competent 
cell method. The use of a plasmid having a genetic marker (e.g., 
antibiotic resistance or the like) as the vector provides a very 
advantageous technique because the selection of transformed Bacillus 
bacteria can be facilitated by utilizing the genetic marker. 
As the vector with which the chromosomal DNA fragments prepared from a 
Bacillus bacterium are linked, plasmids pTP4, pTP5 or pUB110 and phages 
.phi.11, .phi.105 or .phi.1 are preferred. For example, the use of pUB110 
permits the selection of transformants on the basis of kanamycin 
resistance, so that Bacillus bacteria having the plasmid introduced 
thereinto can be efficiently selected on a kanamycin-containing agar 
medium. 
In the resulting large number of transformed Bacillus bacteria, recombinant 
plasmid DNAs including chromosomal DNA fragments having various molecular 
weights are present. Where transformation has been effected by using a 
plasmid as the vector, transformants having increased amounts of secreted 
protein are selected and cultured. Then, the cells are collected and 
plasmid DNA is extracted therefrom according to a conventional procedure. 
Thus, the DNA base sequence of the present invention is included in the 
extracted recombinant plasmid DNA. Transformants having increased amounts 
of secreted protein may be selected by culturing each transformant and 
examining whether or not the amount of protein secreted in the culture 
medium is increased as compared with the host bacterium. However, the 
selection can be facilitated by paying attention to a secreted protein 
having enzyme activity and detecting an increase in the enzyme activity of 
the enzyme protein secreted out of the cells. Such enzymes include, for 
example, amylase, protease, penicillinase, cellulase and alkaline 
phosphatase. Among these enzymes, protease is preferred as an index for 
the selection of transformants because its enzyme activity can be very 
easily determined by measuring the size of the haloes formed on a 
casein-containing agar plate or by enzymatic reaction using casein as the 
substrate. 
Once a transformant having increased amounts of secreted protein has been 
obtained, the capability of its recombinant plasmid in increasing the 
amount of protein secreted by microorganisms can be confirmed by 
extracting plasmid DNA from its cells and introducing the extracted 
plasmid DNA again into the Bacillus bacterium used as the host. Where 
protease is selected as the secreted protein, it is possible to select a 
suitable transformant by taking an increase in extracellular protease 
activity as an index, extract recombinant plasmid DNA from its cells, and 
use the extracted recombinant plasmid DNA for purposes of transformation. 
If all of the transformants thus obtained shown an increase in 
extracellular protease activity, the extracted recombinant plasmid DNA is 
found to include a DNA base sequence capable of increasing the amount of 
protein secreted by microorganisms. Then, the restriction enzyme cleavage 
map of this recombinant plasmid DNA is made to characterize the 
contemplated recombinant plasmid and, on the basis of this cleavage map, 
the DNA base sequence of the inserted chromosomal DNA fragment derived 
from the Bacillus bacterium is determined, for example, by the 
Maxam-Gilbert method [Proc. Natl. Acad-Sci. U.S.A., 74, 560(1977)]. The 
DNA base sequence of the present invention may be obtained as the whole or 
a part of the cloned DNA. If DNA capable of increasing the amount of 
protein secreted by microorganisms is cloned from mutant strains of the 
genus Bacillus or microorganisms of different genera or species, it is 
natural that there may be obtained DNA fragments comprising derivative 
sequences in which the DNA base sequence of the present invention has 
undergone mutational changes such as base substitution, deletion, 
insertion and/or transposition. 
Moreover, microorganisms having very high amounts of secreted protein can 
be bred by incorporating the DNA base sequence of the present invention in 
the chromosomal DNA of a microorganism, particularly a bacterium of the 
genus Bacillus, or a vector and then introducing the resulting recombinant 
DNA into microorganisms. Thus, the DNA base sequence of the present 
invention has very wide applications in the microbial industry. 
The present invention is further illustrated by the following example. 
However, this example is not to be construed to limit the scope of the 
invention. 
EXAMPLE 
Using 2 liters of the NB medium (containing 0.8% meat extract, 0.8% 
polypeptone and 0.4% NaCl and adjusted to pH 7.2), Bacillus 
amyloliquefaciens strain F (ATCC 23350) having high amounts of secretions 
of such proteins as amylase or protease was shake cultured at 37.degree. 
C. The cells were collected in a late phase of logarithmic growth and 
chromosomal DNA was isolated therefrom according to Marmur's method [J. 
Mol. Biol., 3, 208 (1961)]. 
50 .mu.g of this chromosomal DNA was treated with the restriction enzyme 
EcoRI by incubating it in 400 .mu.l of a reaction mixture [containing 100 
mM Tris-HCl (pH 7.5), 7 mM MgCl.sub.2, 50 mM NaCl, 7 mM 2-mercaptoethanol, 
0.01% bovine serum albumin and 20 units of EcoRI (manufactured by Takara 
Shuzo Co.)] at 37.degree. C. for 3 hours. This reaction mixture was 
deprotenized with phenol and, thereafter, the degradation product was 
recovered by precipitation with ethanol. 
Separately, 50 .mu.g of pUB110 DNA, which is a plasmid capable of 
multiplying within the cells of a Bacillus bacterium, was treated in the 
same manner to obtain the complete degradation product of pUB110 DNA 
cleaved by EcoRI. Then, the resulting straight-chain DNA was treated with 
bacterial alkaline phosphatase (hereinafter referred to as "BAP") by 
incubating it in 300 .mu.l of a reaction mixture [containing 50 mM 
Tris-HCl (pH 8.40 and 8 units of BAP (manufactured by Worthington Co.)] at 
65.degree. C. for 4 hours. This reaction mixture was deproteinized with 
phenol and, thereafter, the degradation product of DNA was recovered by 
precipitation with ethanol. 
Using 0.03 unit of T.sub.4 DNA ligase (manufactured by Takara Shuzo Co.), 
0.5 .mu.g of the above pUB110 DNA (EcoRI-cleaved, BAP-treated) and 1 .mu.g 
of the above EcoRI degradation product of chromosomal DNA were linked 
together by incubating them in a reaction mixture [containing 66 mM 
Tris-HCl (pH 7.6), 6.6 mM MgCl.sub.2, 10 mM dithiothreitol and 1 mA ATP] 
at 15.degree. C. for 4 hours. Then, using the method of Chang et al. [Mol. 
Gen. Genet., 168, 111(1978)], the resulting recombinant plasmid was 
introduced into Bacillus subtilis strain 1A289 (aro1906 metB5 sacA321 
amyE) having low protease-secreting ability (a stock strain maintained in 
the Bacillus Genetic Stock Center, the Ohio State University, Columbus, 
Ohio 4329, U.S.A.). 
About 7,500 transformants having kanamycin resistance (Km.sup.4) induced by 
the introduction of the recombinant plasmid into Bacillus subtilis strain 
1A289 were inoculated on an agar medium containing 0.6% casein and 
incubated at 37.degree. C. for 18 hours. Thereafter, transformants having 
increased amounts of secreted protease were selected on the basis of their 
character of forming a large halo around the colony. One of these 
transformants having amounts of secreted protease was cultured in a 
kanamycin-containing medium. Then, recombinant plasmid DNA was extracted 
from its cells according to the method of Birnoboim et al. [Nucleic Acid 
Res., 7, 1513(1979)] and its restriction enzyme cleavage map was made. 
This plasmid was named pNP718 (see FIG. 1). 
Using the aforesaid method of Chang et al., plasmid pNP718 was introduced 
into Bacillus subtilis strain 1A20 (dnaC ilvA1 met B5) having low amounts 
of secreted protease (a stock strain maintained in the Bacillus Genetic 
Stock Center, the Ohio State University, Columbus, Ohio 4329, U.S.A.). As 
a result, all of the resulting kanamycin-resistant transformants exhibited 
high amounts of secreted protease. Using the BY medium (containing 0.5% 
meat extract, 1% polypeptone, 0.2% yeast extract and 0.2% NaCl and 
adjusted to pH 7.2), Bacillus subtilis strain 1A20 used as the host and 
Bacillus subtilis strain 1A20 carrying pNP718 were shake cultured at 
37.degree. C. for 10 hours. Thereafter, each culture medium was filtered 
and the protease activity of the filtrate was determined. The results 
obtained by comparing the extracellular protease activities of these 
strains are shown in Table 1. In the determination of protease activity, 
neutral protease and alkaline protease activities were measured according 
to the method of Hagiwara et al. [J. Biochem., 45, 185(1958)] and 
expressed as relative values. 
TABLE 1 
______________________________________ 
Extracellular 
protease activity 
(relative value) 
Neutral Alkaline 
Strain protease protease 
______________________________________ 
Bacillus subtilis 1A20 
100 100 
Bacillus subtilis lA20pNP718 
1,040 450 
______________________________________ 
Plasmid pNP718 has four ClaI cleavage sites. Accordingly, the inventors 
obtained the partial degradation products of pNP718 cleaved by ClaI and 
recombined them by the use of T.sub.4 DNA ligase to create a smaller 
recombinant plasmid including a gene involved in increasing the amount of 
protein secreted by microorganisms. This new recombinant plasmid will be 
described hereinbelow. Using the aforesaid method of Chang et al., the 
plasmid DNA (mixture) obtained by recombining the partial degradation 
products of pNP718 cleaved by ClaI was introduced into Bacillus subtilis 
strain 1A289. Then, using the aforesaid method of Birnoboim et al., 
plasmid DNA was extracted from some of the resulting transformants. Thus, 
there was obtained plasmid pNP181 having inserted therein a ClaI 1.75 kb 
fragment derived from pNP718 (see FIG. 2). 
Using the method of Chang et al., this plasmid pNP181 was introduced again 
into Bacillus subtilis strain 1A289. When the amount of secreted protease 
of the resulting transformant MT-0181 (FERM BP-343) (a stock strain 
maintained in the Fermentation Research Institute Agency of Industrial 
Science and Technology, 1-3, Higashi 1 chome Yatabe-machi Tsukuba-gun, 
Ibaraki-ken 305, Japan) was determined by measuring the size of the haloes 
formed on the aforesaid casein-containing agar medium, it was confirmed 
that plasmid pNP181 was capable of increasing the amount of protease 
secretions of the bacterium. 
Then, plasmid pNP181 was introduced into the aforesaid Bacillus subtilis 
strain 1A20 having low amount of secreted protease. The resulting 
transformant was examined for any increase in amount of secreted protease 
and the results thus obtained are shown in Table 2. The protease activity 
was determined in the same manner as for Table 1. 
TABLE 2 
______________________________________ 
Extracellular 
protease activity 
(relative value) 
Neutral Alkaline 
Strain protease protease 
______________________________________ 
Bacillus subtilis 1A20 
100 100 
Bacillus subtilis 1A20pNP181 
700 420 
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The ability of this recombinant plasmid pNP181 to promote the secretion of 
neutral protease, alkaline protease and other proteins could also be 
confirmed by subjecting the proteins secreted in the culture medium of the 
transformant to polyacrylamide gel electrophoresis. 
In order to determine the DNA base sequence of the ClaI 1.75 kb fragment, 
an EcoRI 4.0 kb fragment including the ClaI 1.75 kb fragment was cut out 
of plasmid pNP718 and linked with the EcoRI site of Escherichia coli 
vector pBR322 by means of T.sub.4 DNA ligase to prepare recombinant 
plasmid pNP626 (see FIG. 3). 
After plasmid pNP626 DNA was multiplied in Escherichia coli and its copy 
number was increased by means of chloramphenicol, plasmid DNA was 
extracted from the cells. The ClaI 1.75 kb fragment of the extracted 
plasmid DNA was further cleaved by the restriction enzymes HinfI, Sau3A, 
HpaII and TaqI to obtain smaller DNA fragments. The DNA base sequences of 
these smaller DNA fragments were determined according to the Maxam-Gilbert 
method [Proc. Natl. Acad. Sci. U.S.A., 74, 560(1977)]. 
In the ClaI 1.76 kb fragment, one of the strands was found to comprise the 
DNA base sequence claimed in claim 1. As shown in FIG. 4, this DNA base 
sequence includes base sequences (-35 region, TATA box) characteristic of 
the RNA synthesis initiation sites and base sequences (2 stem structures 
and a succeeding T-rich sequence) characteristic of the RNA synthesis 
termination sites. Thus, the presence of base sequences corresponding to 
the initiation sites, the termination sites and the regions located 
therebetween permits the expression of the character of increasing the 
amount of protein secretions of microorganisms. For this reason, the term 
"a part of said DNA base sequence" as used in claim 5 means at least one 
set of base sequences corresponding to an RNA synthesis initiation site, 
an RNA synthesis termination site and the region located therebetween, as 
shown in FIG. 4, or any combination of base sequences including such base 
sequences. In the recombinant plasmids pNP718, pNP181 and pNP626, the DNA 
base sequence claimed in claim 1 is included to result in the expression 
of the aforesaid character. Thus, it is to be understood that these 
recombinant plasmids fall within the scope of the recombinant plasmid 
claimed in claim 5.