Microorganism and process for preparing D-biotin using the same

A microorganism derived from a host microorganism capable of producing d-biotin by introducing a recombinant plasmid being incorporated with a biotin gene cloned from a microorganism of the genus Serratia capable of producing d-biotin and further integrating an exogenous biotin gene into the chromosome, and a process for preparing d-biotin which comprises cultivating the microorganism in a culture medium so that d-biotin is formed and accumulated in the culture medium and collecting the d-biotin. The microorganism of the invention has an extremely high productivity of d-biotin, and hence, d-biotin can be produced in a large amount by cultivating the microorganism of the invention.

The present invention relates to a novel microorganism and a process for 
preparing d-biotin using the same. 
PRIOR ART 
d-Biotin is a vitamin indispensable to human beings or other animals and 
used as a raw material of drugs or as feed additives. As a process for 
preparing d-biotin by a fermentation method using a conventional 
fermentation medium, there is known a method using a 
genetically-engineered microorganism derived from the genus Escherichia or 
Serratia [cf. Japanese Patent Publication (Kohyo) No. 500081/1989, 
Japanese Patent First Publication (Kokai) No. 155081/1987 and Japanese 
Patent First Publication (Kokai) No. 27980/1990]. 
However, the production efficiency of d-biotin in these methods using the 
genetically-engineered microorganism is not so high, and hence, it has 
been required to construct a new microorganism having higher productivity 
of d-biotin and to develop an improved industrial fermentation method 
which can produce d-biotin with sufficiently high production efficiency 
using said microorganism. 
As a result of various investigations, the present inventors have found 
that (i) a mutant microorganism obtained by mutating an actithiazic 
acid-resistant mutant so as to acquire a resistance against ethionine or 
S-aminoethylcysteine shows an increased productivity of d-biotin; (ii) a 
microorganism of the genus Serratia capable of producing d-biotin, wherein 
a fragment of chromosomal deoxyribonucleic acid (hereinafter referred to 
as "DNA") in charge of d-biotin production cloned from a microorganism of 
the genus Serratia is integrated into the chromosome by utilizing a 
transposon, shows an extremely increased productivity of d-biotin; (iii) 
when these microorganisms having higher productivity of d-biotin acquire 
an additional resistance against an acetic acid analogue such as 
chloroacetic acid, such microorganisms can show further extremely 
increased productivity of d-biotin; and further (iv) when the above 
microorganism (ii) or (iii) is incorporated with a recombinant plasmid 
comprising a vector and a biotin gene fragment cloned from an actithiazic 
acid-resistant mutant, the resultant microorganism shows a drastically 
increased productivity of d-biotin, and that d-biotin can advantageously 
be produced on an industrial scale by utilizing this microorganism (iv). 
SUMMARY DESCRIPTION OF THE INVENTION 
An object of the invention is to provide a microorganism derived from a 
host microorganism capable of producing d-biotin by introducing a 
recombinant plasmid being incorporated with a biotin gene cloned from a 
microorganism of the genus Serratia capable of producing d-biotin and 
further by integrating an exogenous biotin gene into the chromosome of 
said host microorganism. 
Another object of the invention is to provide a process for preparing 
d-biotin which comprises cultivating said microorganism in a culture 
medium so that d-biotin is formed and accumulated in the culture medium 
and collecting the produced d-biotin.

DETAILED DESCRIPTION OF THE INVENTION 
[A] Source of Biotin gene 
A biotin gene referred to herein means any gene participating in the 
d-biotin production in vivo such as a gene encoding 7,8-diaminopelargonic 
acid aminotransferase (bioA), a gene encoding biotin synthetase (bioB), a 
gene encoding 7-keto-8-aminopelargonic acid synthetase (bioF), a gene 
encoding pimeloyl-CoA synthetase (bioC) and a gene encoding dethiobiotin 
synthetase (bioD), or a partial portion of these genes. A source of such 
genes includes microorganisms belonging to the genus Serratia [cf. 
Bergey's Manual of Systematic Bacteriology, Vol. 1, page 477 (1984)] which 
are capable of producing d-biotin. The biotin gene used in the present 
invention is preferably derived from a microorganism of the genus Serratia 
showing resistance against biotin analogues such as actithiazic acid, 
5-(2-thienyl)valetic acid, dehydrobiotin, etc. 
Such microorganisms include microorganisms showing actithiazic acid 
resistance, such as Serratia marcescens SB303 (FERM P-10119) and Serratia 
marcescens SB411 [cf. Japanese Patent First Publication (Kokai) No. 
27980/1990]. In addition, microorganisms which do not show any resistance 
against biotin analogues such as a wild strain Serratia marcescens Sr41 
8000 (FERM BP-487) can also be suitably used as a source of the biotin 
gene after making them acquire a resistance against biotin analogues by a 
known method as described in Japanese Patent First Publication (Kokai) No. 
27980/1990. 
There can also be used as a source of the biotin gene those microorganisms 
which, in addition to the resistance against biotin analogues, further 
show resistance against methionine analogues such as ethionine, resistance 
against lysine analogues such as S-aminoethyl-cysteine, and resistance 
against acetic acid analogues such as chloroacetic acid. 
[B] Vector Plasmid 
The vector plasmid used for constructing the recombinant plasmid together 
with the above biotin gene may be any plasmid which is replicable in 
transformed cells, but is preferably plasmids which have a copy number of 
1 to several thousands and contain a resistance marker against an 
antibiotic such as ampicillin, kanamycin, chloramphenicol, etc., and 
further contain an appropriate promoter such as lac, tac, or trp. 
Moreover, the vector plasmids may further contain a plasmid stabilizing 
gene such as par and parB. 
These vector plasmids include, for example, pLG339 [cf. Gene, Vol. 18, page 
335 (1982)], pBR322 [cf. Gene, Vol. 2, page 95 (1977)], pUC18 [cf. Gene, 
Vol. 33, page 103 (1985)], pUC19 [cf. Gene, Vol. 33, page 103 (1985)], 
pHSG298 [cf. Gene, Vol. 61, page 63 (1987)], pHSG299 [cf. Gene, Vol. 61, 
page 63 (1987)], pKG1022 [cf. Biotechnology, Vol. 6, page 1402 (1988)], 
pKT240 [cf. Gene, Vol. 26, page 273 (1983)], and the like. Another 
suitable example of a vector plasmid is a plasmid pCHR81 which is a 
temperature-sensitive replicable plasmid derived from conjugative plasmid 
R388 wherein Tn5 is incorporated as a transposon and a kanamycin-resistant 
gene and a trimethoprim-resistant gene are contained as selection markers 
[cf. Gene, Vol. 56, page 283 (1987)]. 
The above-mentioned plasmids are commercially available, or may be prepared 
from microbial cells containing these plasmids by a conventional method, 
for example, by "cleared lysate method" (cf. Yasuyuki Takagi, "Procedure 
for Experiment in Genetic Engineering", page 125, published by Kodansha, 
1980), or by "alkaline lysis method" [cf. Maniatis et al., "Molecular 
Cloning", page 368, Cold Spring Harbor Laboratory, U.S.A. (1982)]. 
[C] Host Microorganism 
The host microorganism of the present invention may be any microorganism 
capable of producing d-biotin. 
Particularly, preferable host microorganism is microorganisms of the genus 
Serratia having resistance against all of or any one of biotin analogues 
such as actithiazic acid, 5-(2-thienyl)valeric acid, dehydrobiotin, etc., 
acetic acid analogues such as chloroacetic acid, etc., methionine or 
methionine analogues such as ethionine, etc., and lysine analogues such as 
S-aminoethylcysteine, etc. Most preferable one is Serratia marcescens 
having resistances against actithiazic acid, ethionine and 
S-aminoethylcysteine or the Serratia marcescens which further has 
resistance against chloroacetic acid. 
Such host microorganisms include, for example, Serratia marcescens having 
resistance against actithiazic acid and being capable of producing 
d-biotin, such as Serratia marcescens SB303 (FERM P-10119) and Serratia 
marcescens SB411 [Japanese Patent First Publication (Kokai) No. 
27980/1990], Serratia marcescens TA5023 (FERM BP-4101) which contains a 
recombinant plasmid pLGM201 constructed by inserting a biotin gene into a 
vector pLG339, and the like. In addition, there can also suitably be used 
a wild strain Serratia marcescens Sr41 8000 (FERM BP-487) after making it 
acquire resistance against the above substances. 
The above-mentioned resistances can be given to the host microorganisms by 
a known method. For example, an appropriate microorganism is subjected to 
a conventional mutagenic treatment and then cultivated on a minimum agar 
medium supplemented with the above substances against which said 
microorganism is to acquire the desired resistance, such as biotin 
analogues, acetic acid analogues, etc., for example, Davis-Mingioli 
minimum medium [Journal of Bacteriology, Vol. 60, page 17 (1950)] or a 
modified medium thereof wherein the carbon source is replaced with various 
sugars, organic acids or amino acids, at 27.degree. to 37.degree. C. for 3 
to 7 days and the formed large colonies are isolated. Then, the isolated 
microorganisms are tested for their productivity of d-biotin in the 
fermentation culture medium by a microorganism quantification method 
utilizing Lactobacillus plantarum (cf. Seikagaku Jikken Koza, Vol. 13, 
page 355) to select a microorganism having the desired resistance. 
The treatment for giving the resistance to the host microorganism may be 
done either after or before the introduction of a recombinant plasmid or 
integration of an exogenous biotin gene into the chromosome of the host 
microorganism. 
[D] Preparation of Recombinant Plasmid 
The biotin gene can be prepared from the above microorganisms containing 
the biotin gene by a conventional method, for example, by "cleared lysate 
method" [cf. Yasuyuki Takagi, "Procedure for Experiment in Genetic 
Engineering", page 125, published by Kodansha, 1980; Maniatis et al., 
"Molecular Cloning", page 86, Cold Spring Harbor Laboratory, U.S.A. 
(1982)]. For example, a chromosome containing the biotin gene is digested 
with an appropriate restriction enzyme and the obtained gene fragments are 
subjected to an agarose gel electrophoresis. Then, a fraction containing 
the biotin gene fragment is separated from the agarose gel and subjected 
to an electrophoretic elution method [cf. Maniatis et al., "Molecular 
Cloning", page 164, Cold Spring Harbor Laboratory (1982)] using a dialysis 
tube. 
Thereafter, a vector plasmid is partially digested with an appropriate 
restriction enzyme (e.g. EcoRI, HindIII, BamHI, SalI, etc.) and the 
obtained partially digested fragment is ligated to the above biotin gene 
fragment by a DNA ligase (e.g. T4 ligase, E. coli DNA ligase, etc.) to 
prepare a recombinant plasmid. The vector plasmid may be a plasmid 
incorporated with a DNA fragment in charge of plasmid stabilizing gene 
such as parB [Journal of Molecular Biology, Vol. 203, page 119 (1988)] or 
a plasmid containing an appropriate selection marker gene. Then, a 
restriction enzyme-deficient d-biotin-requiring mutant [e.g. Escherichia 
coli .chi.1776 strain (ATCC31244); Molecular Cloning of Recombinant DNA, 
page 248, ed. by Scott and Werner, Academic Press (1977), etc.] is 
transformed with the above-obtained recombinant plasmid DNA and those 
microorganisms which are resistant against kanamycin and do not require 
d-biotin on a culture medium supplemented with 7-keto-8-aminopelargonic 
acid are isolated, followed by extraction of a plasmid DNA from the 
obtained clone to give the desired recombinant plasmid. 
[E] Host Microorganism Wherein Biotin Gene is Incorporated into Chromosome 
A host microorganism wherein a biotin gene is incorporated into the 
chromosome can be prepared by ligating an exogenous biotin gene to a 
plasmid which contains a transposon and a marker gene and introducing said 
plasmid into the host microorganism. 
The plasmid includes pCHR81 plasmid which is constructed for integrating a 
transposon into a chromosome [Gene, Vol. 56, page 283 (1987)]. The plasmid 
pCHR81 contains an incorporated transposon Tn5 and has a 
kanamycin-resistant gene and trimethoprim-resistant gene as selection 
marker genes. The exogenous gene is ligated to an appropriate portion of 
Tn5 utilizing a restriction enzyme site such as SmaI, SalI, BamHI, etc. 
and the resulting recombinant plasmid is then introduced into the host 
microorganism. 
Before introducing the recombinant plasmid into the host microorganism, the 
plasmid wherein the exogenous gene is ligated is preferably modified by a 
modification system of microorganisms of the genus Serratia. For example, 
such a modification can be conducted by introducing the plasmid wherein 
the exogenous gene is ligated into Serratia marcescens TT392 (a mutant 
strain of Serratia marcescens Sr41) [Journal of Bacteriology, Vol. 161, 
page 1 (1985)] by the method of Takagi and Kisumi [Journal of 
Bacteriology, Vol. 161, page 1 (1985)] and isolating the plasmid from the 
obtained transformed microorganism by the cleared lysate method. 
The modified plasmid is then introduced into a host microorganism by the 
method of Takagi and Kisumi and the host microorganism is cultivated at 
about 37.degree. C. for several hours and the obtained culture solution, 
after being appropriately diluted, is applied onto a nutrient agar plate. 
Then, cultivation is done at 30.degree. C. overnight to form colonies and 
those colonies showing kanamycin resistance and trimethoprim sensitivity 
are selected. After the colonies are isolated, the chromosomal DNA from 
the microbial cells is subjected to Southern blotting analysis using a 
probe corresponding to an appropriate portion within Tn5 DNA and thereby 
the strain showing a DNA band at the site of predicted nucleotide number 
can be identified as the host microorganism of the genus Serratia wherein 
the exogenous biotin gene is integrated into the chromosome. 
[F] Preparation of Microorganism of the Genus Serratia Containing a 
Recombinant Plasmid Which Contains a Cloned Biotin Gene Fragment 
The recombinant plasmid constructed by incorporating a biotin gene into a 
vector plasmid can be introduced into the host microorganism wherein a 
biotin gene is incorporated into the chromosome, for example, by the 
method of Takagi and Kisumi. The transformed microorganism can be selected 
by the drug-resistance marker of the recombinant plasmid. The recombinant 
microorganism thus obtained includes, for example, Serratia marcescens 
TA5027 (FERM BP-4103) which is prepared by introducing a recombinant 
plasmid pLGM201PHcA containing a d-biotin gene fragment into a host 
microorganism Serratia marcescens K8CL48 which has resistances against 
actithiazic acid, ethionine, S-aminoethylcysteine and chloroacetic acid 
and contains an exogenous d-biotin gene fragment in the chromosome; 
Serratia marcescens TA5026 (FERM BP-4102) which is prepared by introducing 
a recombinant plasmid pLGM201PHcA containing a d-biotin gene fragment into 
a host microorganism Serratia marcescens ETA23-K8 which has resistances 
against actithiazic acid, ethionine and S-amino-ethylcysteine and contains 
an exogenous d-biotin gene fragment in the chromosome; and the like. 
[G] Cultivation 
The thus prepared microorganism capable of producing d-biotin is cultivated 
so that d-biotin is formed and accumulated in the culture medium at a high 
concentration. 
The medium used for the production of d-biotin includes any conventional 
medium wherein the microorganism can grow. Suitable medium contains a 
carbon source such as saccharides (e.g. glucose, sucrose, molasses, etc.), 
organic acids (e.g. fumaric acid, citric acid, etc.), or alcohols (e.g. 
glycerol, etc.); a nitrogen source such as inorganic ammonium salts (e.g. 
ammonium sulfate, ammonium chloride, etc.) or urea; and an organic 
nutrient such as corn steep liquor, peptone, yeast extract, or casein 
hydrolysate, and the like. The carbon source is usually contained in an 
amount of 10 to 30% by weight based on the whole weight of the medium, the 
nitrogen source is usually contained in an amount of 1 to 3% by weight 
based on the whole weight of the medium, and the organic nutrient is 
usually contained in an amount of 0 to 1% by weight based on the whole 
weight of the medium. The medium may further optionally contain a slight 
amount of potassium phosphate, magnesium sulfate, ferrous sulfate, sodium 
molybdate, etc. It may further optionally contain calcium carbonate, 
ammonium, etc. in order to adjust pH of the medium in a range of 6 to 8. 
The microorganism of the present invention is inoculated into the above 
medium and cultivated by shaking culture at 25.degree. to 37.degree. C. or 
culture under aerobic conditions (e.g. aeration culture) for 3 to 7 days 
and thereby a significant amount of d-biotin can be formed and accumulated 
in the medium. The amount of d-biotin formed and accumulated in the medium 
can further be increased by adding the above medium components to the 
medium at a suitable time or continuously during cultivation. 
[H] Isolation and Purification of D-biotin from Culture Solution 
From the thus obtained d-biotin containing culture solution, d-biotin can 
be isolated and purified in the following manner. 
First, the solution after fermentation is acidified with hydrochloric acid 
and then the microbial cells are removed with a microfilter of hollow 
fiber type. The resultant filtrate is passed through a column filled with 
a high porous type synthetic adsorbent to adsorb d-biotin in the filtrate 
to the adsorbent. After the column is washed with a diluted hydrochloric 
acid solution, the desired d-biotin is eluted by passing methanol--ammonia 
solution (1:9) and the like through the column. 
Then, the eluate containing d-biotin is concentrated under reduced pressure 
and the concentrate is passed through a column filled with a strongly 
basic anion exchange resin to adsorb d-biotin to the resin. The column is 
washed with water and then d-biotin is eluted with an aqueous ammonia and 
the like. The eluate is again concentrated under reduced pressure and is 
adjusted to around pH 3.0 with hydrochloric acid and the like. The 
concentrate is cooled at 5.degree. to 10.degree. C. and allowed to stand 
overnight, thereby d-biotin is crystallized. The obtained crystals are 
then separated by filtration and dried to give d-biotin in crystalline 
form at a high yield. 
EXAMPLES 
The present invention is illustrated by the following Examples but should 
not be construed to be limited thereto. 
In the Examples, d-biotin was measured by the microorganism quantification 
method utilizing Lactobacillus plantarum [cf. Seikagaku Jikken Koza, Vol. 
13, page 355; ed. by Zenji Nose et al., published by Tokyo Kagaku Dojin 
(1975)]. 
Example 1 
(1) Preparation of Host Microorganism Having Resistance Against Actithiazic 
Acid, Ethionine and S-aminoethylcysteine 
(A): Cells of Serratia marcescens SB411 which has resistance against 
actithiazic acid and is capable of producing d-biotin (Japanese Patent 
First Publication (Kokai) No. 27980/1990) are subjected to a mutagenesis 
treatment by the method of Edelberg et al. [Biochemical and Biophysical 
Research Communications, Vol. 18, page 788 (1965)] and cultivated in a 
nutrient culture medium (glucose 0.5%, peptone 1.0%, meat extract 0.3%, 
yeast extract 1.0%, sodium chloride 0.5%) for 1 hour. The culture solution 
is centrifuged (1,000.times.g) and the obtained cells are washed three 
times with physiological saline by centrifugation. The cells are suspended 
in physiological saline and applied onto a minimum agar plate (glucose 
0.5%, potassium dihydrogen phosphate 0.3%, dipotassium hydrogen phosphate 
0.7%, magnesium sulfate 7 hydrate 0.01%, agar 1.5%) containing 60 mM 
DL-ethionine at 1 to 10.times.10.sup.5 cells per plate. After cultivation 
at 30.degree. C. for 5 days, formed large colonies are isolated to give 
ethionine-resistant strains. 
(B): Then, the obtained resistant strains are tested for their productivity 
of d-biotin in a fermentation medium (sucrose 10%, urea 1%, dipotassium 
hydrogen phosphate 0.1%, magnesium sulfate 7 hydrate 0.1%, ferrous sulfate 
7 hydrate 0.01%, calcium carbonate 2%) by the method described in Japanese 
Patent First Publication No. 27980/1990 to give a strain Serratia 
marcescens ET2 which shows a significantly increased productivity of 
d-biotin as compared to the parent strain. 
The amount of d-biotin produced by this strain was 24 mg/l while the amount 
of d-biotin produced by the parent strain was 16 mg/l. 
(C): The obtained Serratia marcescens ET2 cells are subjected to 
mutagenesis treatment in the same manner as in the above process (A) and 
then applied onto a minimum agar plate containing 50 mM 
S-aminoethylcysteine at 1 to 10.times.10.sup.5 cells/plate. After 
cultivation at 30.degree. C. for 5 days, formed large colonies are 
isolated to give S-aminoethylcysteine-resistant strains. These strains are 
tested for their productivity of d-biotin in the fermentation medium as in 
the above process (B) to give a strain Serratia marcescens ETA23 which 
shows a significantly increased productivity of d-biotin as compared to 
the parent strain. 
The amount of d-biotin produced by this strain was 33 mg/l while the amount 
of d-biotin produced by the parent strain was 23 mg/l. 
(2) Integration of Exogenous Biotin Gene into Chromosome of Host 
Microorganism 
(A): Escherichia coli MC1061 containing pCHR81 [Gene, Vol. 56, page 283 
(1987)] is inoculated into L-broth (800 ml) containing 0.2% glucose and 
subjected to shaking culture at 30.degree. C. for 16 hours. The cells are 
collected by centrifugation and lysed with lysozyme and sodium lauryl 
sulfate. Then, sodium chloride is added to the lysate at a final 
concentration of 1M and the resultant lysate is subjected to 
centrifugation (100,000.times.g, 30 min). The supernatant is separated and 
is treated with phenol, supplemented with ethanol and subjected to 
centrifugation. The precipitate is dissolved in 10 mM Tris HCl--1 mM 
ethylenediamine tetraacetic acid 2 Na (pH 7.5) and the solution is 
subjected to density gradient centrifugation equilibrated with cesium 
chloride--ethidium bromide (200,000.times.g, 16 hours) to separate and 
purify a plasmid DNA to give pCHR81 DNA (0.3 mg)(FIG. 3). 
(B): A recombinant plasmid pBM201, which is constructed by treating a 
chromosomal DNA isolated from Serratia marcescens SB303 and a vector 
plasmid pBR322 with HindIII and EcoRI and then ligating them together (cf. 
Japanese Patent First Publication No. 27980/1990; Example 2), is isolated 
in the same manner as in the process (2)(A). DNA (0.5 mg) of this plasmid 
is completely digested with restriction endonuclease BamHI and then 
subjected to agarose gel electrophoresis and the gel containing 6 kb DNA 
fragment is separated. This gel is put into a dialysis tube and an 
electroelution is conducted to give 6 kb DNA fragment (100 .mu.g) which 
contains four genes (i.e. bioB, bioF, bioC, bioD genes) in charge of 
d-biotin production. 
(C): pCHR81 DNA (1 .mu.g) obtained in the process (2)(A) is partially 
digested with restriction enzyme BamHI. After heat-treatment for 10 
minutes, the digested DNA is mixed with the biotin gene fragment (1 .mu.g) 
of the process (B), and the mixture is treated with DNA ligase derived 
from T4 phage under usual conditions to ligate DNA chains to prepare a 
recombinant plasmid. Restriction enzyme-deficient d-biotin-requiring 
Escherichia coli .chi.1776 (ATCC31244) is transformed with the obtained 
recombinant plasmid DNA. The obtained transformed cells are applied onto a 
minimum agar plate (glucose 0.5%, ammonium sulfate 0.1%, dipotassium 
hydrogen phosphate 0.7%, potassium dihydrogen phosphate 0.3%, magnesium 
sulfate 7 hydrate 0.01%, diaminopimelic acid 0.01%, thymidine 0.004%, 
vitamin free casein hydrolysate 0.2%, agar 1.5%) containing kanamycin 
sulfate (100 .mu.g/ml) and dl-dethiobiotin (0.2 .mu.g/ml) and cultivated 
at 30.degree. C. for 1 day. Formed colonies which are resistant against 
kanamycin and do not require d-biotin are isolated. Plasmid DNAs extracted 
from 10 colonies are digested with restriction enzyme BamHI and the 
obtained fragments are subjected to agarose gel electrophoresis analysis 
to give a recombinant plasmid pCHRM4304 wherein the 6 kb biotin gene 
fragment is inserted at the BamHI site within Tn5 of pCHR81 (FIG. 4). In 
this recombinant plasmid, the Tn5 fragment is ligated in the same 
direction as that in pCHR81. 
(D): DNA of the recombinant plasmid pCHRM304 obtained in the process (C) is 
incorporated into cells of strain TT392, which is a restriction 
enzyme-deficient strain of Serratia marcescens, by the method of Takagi 
and Kisumi to give transformed cells. After cultivating the obtained 
transformed cells, a plasmid DNA is isolated and purified therefrom by the 
cleared lysate method to give DNA (250 .mu.g) of the recombinant plasmid 
modified by the modification system of Serratia marcescens. Then, this 
recombinant plasmid DNA is incorporated into Serratia marcescens ETA23 
capable of producing d-biotin by the method of Takagi and Kisumi and the 
cells are cultivated at 37.degree. C. for 7 hours. After being diluted 
appropriately, the culture solution is applied to a nutrient agar plate 
supplemented with kanamycin sulfate (50 .mu.g/ml) and is cultivated at 
30.degree. C. overnight to form colonies. The obtained 1500 colonies are 
examined for the presence of kanamycin resistance and trimethoprim 
resistance to select 8 colonies which are resistant against kanamycin and 
sensitive to trimethoprim. 
These transformed strains are isolated and a chromosomal DNA is extracted 
from the cells and digested with each of restriction enzymes, HindIII and 
BamHI. The obtained DNA fragments are subjected to agarose gel 
electro-phoresis and then Southern blotting analysis is conducted using 
the BamHI--SmaI fragment (540 bp) of Tn5 present in pCHR81 as a probe to 
confirm that Tn5 containing the biotin gene fragments (bioB, bioF, bloC, 
bioD genes) is inserted into the chromosome in the transformed cells 
expressing kanamycin resistance and trimethoprim sensitivity. Then, these 
transformed cells are tested for their productivity of d-biotin in a 
fermentation medium (sucrose 10%, urea 1%, dipotassium hydrogen phosphate 
0.1%, magnesium sulfate 7 hydrate 0.1%, ferrous sulfate 7 hydrate 0.01%, 
calcium carbonate 2%) by the method described in Japanese Patent First 
Publication No. 27980/1990, thereby a strain which shows the most 
increased productivity of d-biotin as compared to the parent strain, 
Serratia marcescens ETA23-K8, is obtained. 
The above Southern blotting analysis further confirmed that, in case of the 
HindIII digestion, the transformed cells expressing kanamycin resistance 
and trimethoprim sensitivity showed a DNA band at the same site as that of 
the HindIII fragment of control pCHRM304, and on the other hand, the 
chromosome of the parent strain ETA23 did not show a DNA band reacting 
with the probe. The analysis further confirmed that, in case of the BamHI 
digestion, the transformed cells expressing kanamycin resistance and 
trimethoprim sensitivity showed a DNA band having a size different from 
that of the BamHI fragment of control pCHRM304, and on the other hand, the 
chromosome of the parent strain ETA23 did not show a DNA band reacting 
with the probe. 
(3) Acquisition of Resistance Against Chloroacetic Acid by Host 
Microorganism Wherein an Exogenous Biotin Gene is Integrated into the 
Chromosome 
The Serratia marcescens ETA23-K8 cells obtained in the above process are 
subjected to mutagenesis treatment by the method of Ederberg et al. and 
cultivated in a nutrient medium for 1 hour. The culture solution is 
centrifuged and the obtained cells are washed three times with 
physiological saline by centrifugation. The cells are suspended in 
physiological saline and applied onto a minimum agar plate (L-proline 
0.1%, ammonium sulfate 0.1%, potassium dihydrogen phosphate 0.3%, 
dipotassium hydrogen phosphate 0.7%, magnesium sulfate 7 hydrate 0.01%, 
agar 1.5%) supplemented with chloroacetic acid (1 mg/ml) at 1 to 
10.times.10.sup.5 cells per plate. After cultivation at 30.degree. C. for 
5 days, formed large colonies are isolated to give a chloroacetic 
acid-resistant strain, Serratia marcescens K8CL48. 
The obtained drug-resistant strain was tested for its productivity of 
d-biotin in a fermentation medium (glucose 5%, urea 1%, potassium 
dihydrogen phosphate 0.1%, magnesium sulfate 7 hydrate 0.1%, ferrous 
sulfate 7 hydrate 0.01%, corn steep liquor 0.6%, calcium carbonate 2%), 
and as a result, this strain produced 26 mg/l of d-biotin during 48 hour 
cultivation while the parent strain produced 13 mg/l of d-biotin. 
(4) Preparation of Recombinant Plasmid Containing Biotin Gene Fragment 
Serratia marcescens TA5023 (FERM BP-4101) is treated in the same manner as 
in the process (2)(A) to give DNA of pLGM201 having a kanamycin-resistant 
gene as a selection marker wherein the biotin gene fragment (bioA, bioB, 
bioC, bioD genes) derived from Serratia marcescens SB303 is incorporated 
into the EcoRI--HindIII digestion sites of vector pLG339. 
Separately, from Escherichia coli HB101 containing plasmid pKG1022 having a 
plasmid-stabilizing gene parB, DNA (0.3 mg) of pKG1022 is obtained in the 
same manner as in the process (2)(A) and digested with restriction 
endonucleases EcoRI and BamHI. The obtained DNA fragments are subjected to 
agarose gel electrophoresis and a DNA band of 580 bp is cut off from the 
gel and subjected to the electroelution method to give a DNA fragment 
containing parB region. 
The vector pLG339 DNA (0.3 mg) is obtained from Escherichia coli 
C600r.sup.- m.sup.- (ATCC33525) containing the vector pLG339 in the same 
manner as in the process (2)(A). The obtained DNA is digested with 
restriction enzyme HincII and the digested DNA is ligated with the above 
DNA fragment containing parB region, which is blunt-ended with the Klenow 
enzyme, using DNA ligase. Escherichia coli HB101 is transformed with the 
ligated DNA. A clone expressing kanamycin resistance and tetracycline 
sensitivity is selected and analyzed for its restriction enzyme map of the 
plasmid DNA, and thereby a recombinant plasmid pLG339P, wherein parB is 
inserted into the HincII site of pLG339, is obtained. 
The recombinant plasmid pLGM201 DNA is completely digested with EcoRI and 
HindIII. On the other hand, pLG339P obtained above is completely digested 
with EcoRI and then partially digested with HindIII. Both digested DNAs of 
pLGM201 and of pLG339P are combined and used for transformation of 
Escherichia coli .chi.1776 (ATCC31244). From transformed cells, a clone 
which is resistant against kanamycin and does not require biotin is 
selected to give a plasmid pLGM201PHc containing the biotin gene fragment 
derived from Serratia marcescens SB303, the plasmid-stabilizing gene and 
the kanamycin-resistant gene wherein parB is inserted into the HincII site 
of pLGM201. 
From Escherichia coli HB101 containing a plasmid pKT240 having an 
ampicillin-resistant gene, pKT240 DNA is obtained in the same manner as in 
the process (2)(A) and digested with BamHI and BstPI. The obtained DNA 
fragments are subjected to agarose gel electrophoresis and a DNA band of 3 
kb is cut off from the gel and subjected to the electroelution method to 
give a DNA fragment containing the ampicillin-resistant gene region. This 
fragment is blunt-ended with the Klenow enzyme. 
On the other hand, the pLGM201PHc DNA obtained above is also digested with 
EcoRI and XhoI and blunt-ended with the Klenow enzyme. This DNA and the 
above blunt-ended DNA fragment containing ampicillin-resistant gene region 
are ligated with DNA ligase. Escherichia coli C600r.sup.- m.sup.- is 
transformed with the ligated DNA to give a recombinant plasmid pLGM201PHcA 
wherein the kanamycin-resistant gene of pLGM201PHc is substituted with the 
ampicillin-resistant gene derived from pKT2401 (cf. FIG. 2). 
(5) Preparation of the Desired Microorganism 
The recombinant plasmid pLGM201PHcA obtained in the above process (4) is 
introduced into a restriction enzyme-deficient strain Serratia marcescens 
TT392 by the method of Takagi and Kisumi to give transformed cells. After 
cultivating the obtained transformed cells, a plasmid DNA is isolated and 
purified from the cells by the cleared lysate method to give a plasmid 
(300 .mu.g) modified by the modification system of Serratia marcescens. 
Then, this plasmid is introduced into Serratia marcescens ETA23K-8 cells 
obtained above by the method of Takagi and Kisumi and the cells are 
applied onto a nutrient agar plate supplemented with ampicillin (500 
.mu.g/ml) and cultivated at 30.degree. C. overnight. Formed colonies are 
isolated from the medium to give Serratia marcescens TA5026 (FERM BP-4102) 
containing the recombinant plasmid pLGM201PHcA wherein the host 
microorganism is Serratia marcescens ETA23K-8 (cf. FIG. 1). 
The plasmid DNA contained in this strain was confirmed to be identical to 
pLGM201PHcA DNA by the analysis of restriction enzyme digestion map. 
Separately, the plasmid pLGM201PHcA is introduced into Serratia marcescens 
KSCL48 cells obtained above by the method of Takagi and Kisumi and the 
cells are applied onto a nutrient agar plate supplemented with ampicillin 
(500 .mu.g/ml) and cultivated at 30.degree. C. overnight. Formed colonies 
are isolated from the plate to give Serratia marcescens TA5027 (FERM 
BP-4103) containing pLGM201PHcA wherein the host microorganism is Serratia 
marcescens KSCL48 (cf. FIG. 1). 
The plasmid DNA contained in this strain was confirmed to be identical to 
pLGM201PHcA DNA by the analysis of restriction enzyme digestion map. 
Example 2 
Serratia marcescens TA5026 obtained in Example 1 is cultivated on an agar 
slant of L-broth containing ampicillin (500 .mu.g/ml) overnight and then a 
loopful of the cultivated cells is inoculated into a fermentation medium 
comprising a sterilized solution (15 ml) containing sucrose 15%, urea 
1.5%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 7 hydrate 
0.2%, ferrous sulfate 7 hydrate 0.01%, corn steep liquor 0.1% and calcium 
carbonate 1% (wherein sucrose is added to the solution after 
sterilization) in a 500 ml shaking flask. Then, a reciprocal shaking 
cultivation (7 cm stroke; 120 r.p.m.) is conducted at 30.degree. C. for 
120 hours. 
The amount of d-biotin produced in the above culture was measured. The 
results are shown in Table 1 wherein control strains are Serratia 
marcescens SB411 (Japanese Patent First Publication (Kokai) No. 
27980/1990) and Serratia marcescens TA5024 (Japanese Patent First 
Publication (Kokai) No. 27980/1990), which was obtained by introducing the 
recombinant plasmid pLGM201 into SB411. 
TABLE 1 
______________________________________ 
Name of microorganism 
Amount of d-biotin produced 
(Serratia marcescens) 
and accumulated (mg/l) 
______________________________________ 
Control 
SB411 30 
TA5024 150 
The TA5026 250 
present 
invention 
______________________________________ 
Example 3 
Each of Serratia marcescens TA5026 and Serratia marcescens TA5027 is 
cultivated in an agar slant of L-broth containing ampicillin (500 
.mu.g/ml) or kanamycin sulfate (100 .mu.g/ml) or in the same agar slant of 
L-broth containing neither ampicillin nor kanamycin sulfate overnight and 
then a loopful of the cultivated cells is inoculated into a fermentation 
medium comprising a sterilized solution (15 ml) containing glucose 7%, 
urea 1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 7 hydrate 
0.1%, ferrous sulfate 7 hydrate 0.01%, corn steep liquor 0.1% and calcium 
carbonate 4% (wherein glucose is added to the solution after 
sterilization) in a 500 ml shaking flask. Then, a reciprocal shaking 
cultivation (7 cm stroke; 120 r.p.m.) is conducted at 30.degree. C. for 
120 hours. 
The amount of d-biotin produced in the above culture was measured. The 
results are shown in Table 2 wherein control strains are Serratia 
marcescens SB411 and Serratia marcescens TA5024. 
TABLE 2 
______________________________________ 
Name of microorganism 
Amount of d-biotin produced 
(Serratia marcescens) 
and accumulated (mg/l) 
______________________________________ 
Control 
SB411 10 
TA5024 54 
The TA5026 70 
present 
TA5027 96 
invention 
______________________________________ 
Example 4 
Serratia marcescens TA5026 obtained in Example 1 is cultivated in an agar 
slant of L-broth containing ampicillin (500 .mu.g/ml) overnight and then a 
loopful of the cultivated cells is inoculated into a preculture medium 
comprising a sterilized solution (30 ml) containing sucrose 10%, urea 1%, 
dipotassium hydrogen phosphate 0.1%, magnesium sulfate 7 hydrate 0.2%, 
ferrous sulfate 7 hydrate 0.01%, corn steep liquor 0.6% and calcium 
carbonate 1% (wherein sucrose is added to the solution after 
sterilization) in a 500 ml shaking flask. Then, a reciprocal shaking 
cultivation (7 cm stroke; 120 r.p.m.) is conducted at 30.degree. C. for 24 
hours. The obtained preculture solution (14 ml) is inoculated into a 
fermentation medium [sucrose 15%, urea 1.5%, dipotassium hydrogen 
phosphate 0.08%, magnesium sulfate 7 hydrate 0.2%, ferrous sulfate 7 
hydrate 0.01%, corn steep liquor 0.1%, calcium carbonate 4.2% and Disfoam 
CA220 (antifoaming agent, manufactured by Nippon Yushi K.K., Japan) 0.28%; 
1.0 liter] and cultivated in a 1.8 liter jar fermenter at 30.degree. C. at 
aeration rate of 0.5 liter/min. while the rate of agitating is regulated 
so that the dissolved oxygen concentration is maintained to 10% of the 
saturated concentration. From 48 to 120 hours after starting cultivation, 
an additional medium prepared separately (sucrose 67.5%, urea 5%, 
dipotassium hydrogen phosphate 0.15% and corn steep liquor 0.1%) is 
continuously added to the medium at the flow rate of 5.6 ml/h. And 
separately, from 48 to 120 hours after starting cultivation, 0.67 ml of 
20% magnesium sulfate 7 hydrate and 0.97 ml of 2% ferrous sulfate 7 
hydrate are added to the medium every 24 hours. The cultivation is 
conducted for 144 hours to give a culture solution (about 1.3 liters) 
containing 540 mg/l of d-biotin. 
Example 5 
Serratia marcescens TA5027 obtained in Example 1 is cultivated in an agar 
slant of L-broth containing ampicillin (500 .mu.g/ml) overnight and then a 
loopful of the cultivated cells is inoculated into a preculture medium 
comprising a sterilized solution (30 ml) containing glucose 5%, urea 1%, 
dipotassium hydrogen phosphate 0.1%, magnesium sulfate 7 hydrate 0.2%, 
ferric sulfate 0.01%, corn steep liquor 0.6% and calcium carbonate 1% 
(wherein glucose is added to the solution after sterilization) in a 500 ml 
shaking flask. Then, a reciprocal shaking cultivation (7 cm stroke; 120 
r.p.m.) is conducted at 30.degree. C. for 24 hours. The obtained 
preculture solution (14 ml) is inoculated into a fermentation medium 
[glucose 5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0.2%, 
magnesium sulfate 7 hydrate 0.2%, ferric sulfate 0.001%, corn steep liquor 
0.7% and Disfoam CA220 (antifoaming agent, manufactured by Nippon Yushi 
K.K., Japan) 0.28% (pH 7.0); 1.0 liter] and cultivated in a 1.8 liter jar 
fermenter at 30.degree. C. at aeration rate of 0.5 liter/min. while the 
rate of agitating is regulated so that the dissolved oxygen concentration 
is maintained to 10% of the saturated concentration. The culture solution 
is regulated to pH 7.4 or more with a mixture of potassium 
hydroxide--ammonia. From 1 to 3 days after starting cultivation, an 
additional medium prepared separately (glucose 57.5%, ammonium sulfate 
0.5%, dipotassium hydrogen phosphate 0.34%, magnesium sulfate 7 hydrate 
0.2%, ferric sulfate 0.01% and corn steep liquor 2.75%) is continuously 
added to the medium at the flow rate of 3 ml/h to 15 ml/h. The cultivation 
is conducted for 3 days to give a culture solution (about 1.3 liters) 
containing 260 mg/l of d-biotin. 
Example 6 
The culture solution (6.0 liters) obtained in accordance with the 
procedures in Example 4 is acidified to pH 2.7 with hydrochloric acid and 
then the cells are removed with a microfilter (Microza SP113, manufactured 
by Asahi Chemical Industry Co., Ltd., Japan) to give a filtrate (7.6 
liters). The obtained filtrate is passed through a column filled with a 
high porous type synthetic adsorbent (SP207, manufactured by Mitsubishi 
Kasei Corporation, Japan)(amount of the resin, 1000 ml; column diameter, 
40 mm) at the flow rate of SV=2 to adsorb d-biotin in the filtrate to the 
adsorbent and then one liter of diluted hydrochloric acid (pH 3.0) is 
passed through the column at the flow rate of SV=2 to wash the adsorbent. 
Then, d-biotin is eluted by passing a methanol--ammonia solution (1:9) 
through the column at the flow rate of SV=2. 
The eluate (1.8 liters) obtained above is concentrated under reduced 
pressure with a rotary evaporator to give a concentrate (900 ml). The 
obtained concentrate is then passed through a column filled with a 
strongly basic anion exchange resin (Diaion SA-11A, manufactured by 
Mitsubishi Kasei Corporation, Japan)(amount of the resin 100 ml; column 
diameter 20 mm) at the flow rate of SV=2 to adsorb d-biotin to the resin. 
The column is washed with water (200 ml) and d-biotin is eluted by passing 
900 ml of 0.2N ammonium chloride--0.2N ammonia (1:1) through the column at 
the flow rate of SV=2. The obtained eluate (900 ml) is concentrated under 
reduced pressure to a volume of 60 ml and the concentrate is adjusted to 
pH 2.7 with conc. hydrochloric acid and allowed to stand at 10.degree. C. 
or below overnight, thereby d-biotin is crystallized. The formed crystals 
are separated by filtration and dried to give crude crystals (4.5 g), 
which are recrystallized from the water solution to give 2.3 g of pure 
d-biotin. 
EFFECTS OF THE INVENTION 
The microorganism of the present invention, i.e. a microorganism derived 
from a host microorganism capable of producing d-biotin by introducing a 
recombinant plasmid being incorporated with a biotin gene cloned from a 
microorganism of the genus Serratia capable of producing d-biotin and 
further by integrating an exogenous biotin gene into the chromosome of 
said host microorganism, has an extremely high productivity of d-biotin, 
and hence, d-biotin can be produced in a large amount by cultivating the 
microorganism of the invention.