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Instability of the mutated biotin operon plasmid in a biotin-producing mutant of Serratia marcescens
Journal of Biotechnology
43 (1995) 11-19
Instability of the mutated biotin operon plasmid in a biotin-producing mutant of Serratia marcescens Naoki Sakurai apbT *, Yuji Imai a, Saburo Komatsubara
ayb
a Research Laboratory ofApplied Biochemistry, Tanabe Seiyaku Co., Ltd., 16-89 Kashima-3-chome, Yodogawa-ku, Osaka 532, Japan b Research Laboratory ofApplied
Biochemistry
at Toda, Tanabe Seiyaku Co., Ltd., 2-50, Kawagishi-2-chome, Japan
Received 8 September
Toda-shi, Saitama 335,
1994; revised 24 April 1995; accepted 22 May 1995
Abstract The growth of a d-biotin-producing strain of Serratia marcescens (SB412) was strongly inhibited by the introduction of pLGM412, a low-copy-number plasmid containing the complete biotin (bio) operon derived from SB412, whereas the wild-type strain was not inhibited by the plasmid. SB412 carrying pLGM412 was genetically unstable; large colonies appeared spontaneously from the background small colonies. When the plasmids from the large colonies were transformed into the SB412 host, all of the resultant transformants showed a large-colony phenotype, suggesting that the large-colony phenotype is due to mutations in the plasmid-born bio genes. Some of these plasmids were structurally altered and the others
were not. Furthermore, the structurally altered plasmids were classified into a deleted and an elongated type. All of the mutated pLGM412 derivatives reduced or lacked the bio gene expression, indicating that the high expression of bio gene(s) causes the growth inhibition. growth inhibition. Keywords:
d-Biotin producing
By subcloning
experiments,
strain; Serratia marcescens;
biotin synthase (the bioB gene product) was responsible
Growth inhibition;
1. Introduction We have been studying d-biotin production by regulatory mutants of Serratia marcescens Sr41 and have shown alterations in d-biotin-mediated feedback repression of biotin biosynthesis in these mutants (Sakurai et al., 1993a). Strain SB304, resistant to a lower concentration of a biotin analog, acidomycin, produced d-biotin at 5 mg 1-l and strain SB412, derived from SB304 and resistant to a
* Corresponding author: Research Laboratory of Applied Biochemistry at Toda, Tanabe Seiyaku Co., Ltd., 2-50, Kawagishi-2chome, Toda-shi, Saitama 335, Japan. 0168-1656/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1656(95)00103-4
Large colony; Mutated
for the
bio gene; Biotin synthase
higher concentration of acidomycin, produced d-biotin at 20 mg 1-l (Sakurai et al., 1993a). Genetic analyses have revealed that the primary and secondary mutations for d-biotin overproduction occurred in the bio genes of SB304 and SB412, respectively. Subsequently, we cloned the mutated bio operon and found that the two strains with the cloned bio operon showed a high d-biotin production (Sakurai et al., 1993b). Strain SB412 harboring plasa low-copy-number plasmid mid pLGM304, (pLG339) containing the mutated bio operon of SB304, produced d-biotin at 200 mg 1-l in a shaking flask. Since SB412 had two mutations in the bio genes
.V. Sakurut et ~1. /.lountul
12
of Biotechnology
cluster for d-biotin overproduction, we expected that the most productive strain would be obtained by transforming SB412 with pLGM412, a pLG339 derivative containing the SB412 hio operon or by the gene dosage effect of the ho genes in a highcopy-number plasmid. However. we failed to obtain such strains because of a strong growth inhibition by these recombinant plasmids. Here we report the growth inhibition of a biotinproducing strain by the mutated hio genes in plasmid.
43 (1995)
I I-l 9
0.5% glucose, 1.0% peptone, 0.3% meat extract, 1.0% yeast extract, and 0.5% NaCl was used for routine colony isolation and growth study. The minimal medium of Davis and Mingioli (1950) was modified by omitting sodium citrate and increasing the glucose concentration to 0.5%. Medium F2, used for d-biotin production, contained 15% sucrose, 1.5% urea, 0.1% corn steep liquor, 0.1% K,HPO,, 0.2% MgSO, .7H,O, 0.01% FeSO, .7H,O, and 1.5% CaCO,. 2.2. d-Biotin production
2. Materials
and methods
2.1. Strains, plasmids,
and media
Bacterial strains and plasmids used in this work are listed in Table I. Nutrient medium containing
Cells were cultured in a shaking flask containing medium F2 as described previously (Sakurai et al.. 1993b). d-Biotin was assayed by the method of Snell et al. (I 940). Biotin-related compounds, which correspond to d-biotin plus dethiobiotin, were assayed as described previously (Sakurai et al., 1993a).
Table I Bacterial strains and plasmids used Strain and plasmid
Characteristic
E. co/r strains HBIOI
”
Ref.
Fan hsaS-70 (I~. mn)
resA13 uru-14proA2
rhi-I lucYI galK2 rpsL20 xyl-5 mtl-I supE44
MALI01 R879 R87S R872 R87h RX77 S. IIIU~C~~~II.Sstrains 8000 TT3’): SB412 SB412RI SB412R2 Plasmids pLG339 pBR327 pBWlO1 pBM412 pLGWlO1 pLGM304 pLGM412 pLGWlOlB pLGM412B
F
J
bid-74 hioBl7 hioF
( gpt-proAB-argF-luc) IN (rmD-rmE) IN ( rmD-rrE)
I
IN (rmD-rrnE)
I
IN CrrnD-r(nE:’
wild type Nut r resistant to 2.0 mg of acidomycin rrtA::Mu dl rccA::Mu dl Km’ Tc’ pur AP’ pBR322::wbio Ap’ pBR322::mbiu,,, Ap’ pLG33Y::wbio Km’ pLG33Y::mbiu,,, Km’ pLG339::mbro,,, Km’ pLG339::wbioB Km’
-
pLG339::mbioB,,,
Km’
I
I
per ml
Bayer and Roulland-Dussoix,
A
XIII rpsL [Mu dl (lx,
I
hioC’IX (~hlAlX IN (rrrtD-rrnE1 hroDl9
leuB6
AP)]
used for plasmid constructions Casadaban and Cohen, 1979 Del Campillo-Campbell et al., Del Campillo-Campbell et al., Del Campillo-Campbell et al., Del Campillo-Campbell et al., Del Campillo-Campbell et al.,
1969;
1967 1967 1967 1967 1967
Matsumoto et al.. 1973 Takagi and Kisumi, 1985 Sakurai et al., 1993a this study (mutant from strain SB412) this study (mutant from strain SB412) Stoker et al., 1982 Bolivcr et al., 1977 Sakurai et al., 1993b Sakurai et al., 1993b Sakurai et al., 1993b Sakurai et al., 1993b Sakurai et al., 1993b this study this study
’ Symbols used for relevant genotypes and phenotypes arc as follows: Nut-, no production of extracellular nuclease; I-, defect of host restriction enzyme; pur. active plasmid partitioning; whio, wild-type biotin operon; mbiojo,, biotin operon allele of SB304; mbio,tz, biotin operon allele of SB412; wbioB. wild-type hi& gene; mbioBj,r. bioB gene of SB412.
N. Sakurai et al./Journal
2.3. Genetic procedures niques
and recombinant
ofBiotechnology 43 (1995) 11-19
of the cell suspension (lo4 to lo5 cells per ml) was spread on nutrient agar plates. After 3 d of incubation at 30°C large colonies found among small colonies were scored. Subsequently, plasmids of large colonies were analyzed.
DNA tech-
Standard procedures were used for plasmid preparation, restriction enzyme digestion, ligation, and agarose gel electrophoresis (Maniatis et al., 1982). Transformation of Escherichia coli and S. marcescens was carried out according Maniatis et al. (1982). 2.4. Analysis
of plasmid
2.5. Curing of the plasmids from large-colony-forming derioatives of SB412cpLGM412) Cells of large-colony-forming derivatives of SB412(pLGM412) were cultured in 3 ml of nutrient medium for 48 h at 30°C with shaking. Cells were diluted with saline and spread onto nutrient agar plates without kanamycin. Colonies formed were tested for the kanamycin sensitivity and the presence of plasmids.
instability
A single small colony of SB412(pLGM412) grown on a nutrient agar plate was inoculated into a test tube containing 3 ml of a nutrient medium. Incubation was continued at 30°C for 24 h with shaking. The culture was diluted with saline. A O.l-ml aliquot Hidll
ECdl
f-------7.2
13
HindIll
kb ____jt
pBM412 (or pBW101) Aval-Stuldigestion
I
Hincll
digestion
HindIll
Fig. 1. Restriction
maps of pLGWlOlB
and pLGM412B
containing
the bioB
genes of the wild strain and SB412, respectively.
IV. Sakurur et al./Journul
I4
ofBiotechnology 43
(1995) II-19
2.6. Plasmid constructions Plasmids pLGW101B and pLGM412B were constructed as follows. The 2.0-kb Al,aI-StuI fragments carrying the bioB gene and its promoter region were excised from pBWlO1 and pBM412, and were ligated into the HiilcII site of pLG339, producing pLGWlOlB and pLGM412B, respectively (Fig. I ). 2.7. Construction
of the recA mutant
Mu dl phage was used to isolate the recA mutant of SB412. Mu dl is a derivative of phage Mu which contains an ampicillin-resistance marker and can insert its DNA into the chromosomes of various bacteria (Van de Patte et al.. 1980). Lysate obtained by heat induction of E. co/i MAL103 was used to create random Mu dl chromosomal insertions in ampicillin-resistant colonies were SB412. First, screened for the recA mutation by measuring their sensitivity to ultraviolet irradiation. Further, the locus destructed by Mu dl were cloned from the candidate mutants by using ampicillin resistance carried by Mu dl as the selective marker. The recA mutations were confirmed by sequencing the cloned genes, and the sequences were compared with that of the S. marcescens recA gene reported by Ball et al. ( 1990). 2.X Chemicals d-Biotin was a product of Tanabe Seiyaku C’o.. Ltd. Dethiobiotin was prepared and given by Production Technology Division of Tanabe Seiyaku Co.. Ltd. Restriction endonucleases and T4 DNA ligase were purchased from Takara Shuzo Co., Ltd., Kyoto, Japan. Other chemicals were also obtained commercially and not purified further.
3. Results and discussion 3.1. Growth inhibition operon plasmid
ofSB412
by the mutated bio
The mutations for d-biotin overproduction occurred in the bio genes of strain SB412 (Sakurai et al., 1993a). To construct a more productive strain,
Fig. 2. Colony sizes of strains 8000 (wild type) and SB412 Cd-biotin-producing mutant) carrying recombinant plasmids of the hio operons. Cells obtained by single colony isolation were grown in a nutrient medium containing kanamycin for 24 h and spread onto nutrient agar plates containing kanamycin. The plates were incubated for 2 d at 30°C except the plates for SB412(pLGM412), which was incubated for 3 d at 30°C. Photographs: (A). 8000(pLGM412); (B), XOOtKpLGM304); CC), 8000(pLG33Y); CD). SB412cpLGM412); (E). SB412(pLGM304); (F), SB412(pLG33Y).
SB412 was transformed with high-copy-number plasmids, pBM304 and pBM412, consisted of a pBR322 replicon and the mutated bio genes of SB304 and SB412, respectively. These two plasmids, however, could not be introduced into SB412 host under various transformation conditions, whereas strain 8000, wild-type of S. marcescens. was transformed with them at high frequencies. Then, WC attempted to introduce pLGM412, a low-copynumber plasmid with SB412 bio genes into SB412. Transformants of SB412 bearing pLGM412 yielded a few large colonies but the majority of colonies were barely visible after incubation for 3 d at 30°C on nutrient plates containing kanamycin (Fig. 3). These small colonies exhibited very poor growth when transferred to fresh kanamycin nutrient plates and grew to very low densities even when incubated for 2 to 3 d in liquid kanamycin nutrient medium. However, all of the transformants showing large-colony phenotype grew fast as did SB412 (Fig. 2). The colonies were uniform in size and similar in size to that of SB412, a host strain, on kanamycin nutrient plates, and grew to high density in kanamycin nutrient liquid medium. Under the conditions, SB412 carrying pLG339 and pLGW101, a pLG339 deriva-
N. Sakurai et al. /Journal
of Biorechnology 43 (1995) 11 -I 9
tive with the wild-type bio genes, exhibited the similar colonies in size. In contrast, the introduction of pLGM412 into strain 8000 (wild strain) did not cause growth inhibition. Thus, the growth inhibition by the introduction of pLGM412 occurred in SB412 background, suggesting that the growth inhibition by pLGM412 might be due to the high expression of the bio gene(s). Under the conditions described above, no large colonies were generated from SB412 carrying pLGM304, although the colony size of this strain was half that of SB412. To examine the profile of the growth inhibition observed in SB412(pLGM412), we analyzed the host physiology and the plasmids from large colonies generated from small colonies.
SB412(pLGWlOl) (Table 21, indicating that mutations for reducing d-biotin production occurred in either host cells or plasmids in the large-colony strains.
3.3. Curing of plasmids from large-colony strains To determine whether the mutation for decreased d-biotin production in large-colony strains resided in the host strain or in the plasmid, plasmids of ten large-colony strains isolated independently were cured of the plasmids and resulting cured strains were tested for d-biotin production. These cured strains accumulated d-biotin at a concentration of about 40 mg 1-l as did SB412. This result suggested that no genetic alterations for the reduction of d-biotin production occurred in the host cells of large-colony strains. Subsequently, we re-transformed the cells of SB412 with plasmid DNAs extracted from each of above ten large-colony strains. d-Biotin production of resulting recombinants was between 40 and 110 mg 1 - ’ (Table 2).
3.2. d-Biotin production by large-colony strains First, we examined d-biotin production by 30 large-colony strains obtained from independent small colonies of SB412(pLGM412). The amounts of dbiotin produced ranged from 40 mg 1-l to 110 mg 1-l. The former value was identical to that of SB412 and the latter value was similar to that of
Table 2 Characteristics
of pLGM412
Derivatives of pLGM412 (type of mutation) a
pLG339 (vector) pLGW101 pLGM304 D-l (deleted) D-2 (deleted) E-l (elongated) E;2 (elongated) P-l (identical) P-2 (identical)
15
derivatives Complementation bioA
bioB
b
Productivity bioF
bioC
_
_e +’ + + + + + +
bioD
1 + _ _ _ + _
1 + + _
1 + + _
+ + +
+ + + +
+ + + +
+ + + +
of SB412 transformants
d-Biotin
Biotin-related
(mg 1-l 1
(mg 1-t)
35 100 190 36 33 32 42 110 36
50 120 310 52 45 48 58 200 53
’
compounds
d
a Derivatives of pLGM412 were extracted from the large colonies of SB412 (pLGM412) and the restriction patterns were examined with 0.7% agarose-gel electrophoresis. b The plasmid DNAs were introduced into E. coli biotin auxotrophs (R879, R875, R872, R876, R877) and the resultant transformants were tested for complementation of Bio- phenotype. ’ The mutant plasmids which are representatives of each type were introduced into SB412. Cells of the re-transformants were grown at 30°C for 120 h in shaking flasks containing medium F2. A reference strain, SB412, produced 40 mg of d-biotin per liter under these conditions. d Total amount of d-biotin and dethiobiotin. e -, no growth. f +, growth.
N. Sakurai et d/Journal
16
3.4. Structural
changes
ofBiotechnology
of the plasmids of large-col-
ony strains
We analyzed restriction maps of the plasmid DNAs extracted from the each of 300 large-colony strains, which were derived from ten independent small colonies, as follows. Plasmid DNAs were prepared from the large-colony strains and were examined by size analysis on agarose gels (Fig. 3A). When pLGM412 was digested with EcoRI and HindIII, 1.9-, 4.4-, and 7.2-kb DNA bands were observed (Fig. 3B). 1.9- and 4.4-kb bands were derived from the vector plasmid, pLG339, and a 7.2-kb fragment contained the bio gene cluster. Fig. 3 shows a typical restriction pattern for 20 plasmids isolated from the large-colony strains derived from a small colony. The 7.2-kb fragment of seven plasmids (lanes 3, 4, 6, 10, 12, 14, and 15; deleted type) were
09
h 12
J3 (1995) 11-19
subject to deletion, in four plasmids (lanes 2, 7, 9, and 11; elongated type) were inserted by a short fragment of about 1 kb, and the rest (lanes 1, 5, 8, 13, 16, 17, 18, 19, and 20; identical type) had no detectable changes in the 7.2-kb fragment. The ratio of three types of the mutant plasmids in the 300 large colonies was 17% for deleted type, 74% for identical type, and 9% for elongated type. All of the deleted-type plasmids were found to lack either the full-length bio operon, a partial region of the bioB gene, or an entire region of the bioB gene. All of the elongated-type plasmids were inserted by an 1-kb additional fragments into the bioB structural gene. The site of the insertion was apparently the same in the independent elongated types as judged by restriction analysis. The origin of the inserted DNA, however, has not been specified yet. Identical-type plasmids were very similar to pLGM412 in the restric-
3 4 5 6 7 8 9ltlIlPl314l5l617BlPzIC
-7.2 -
kb 4.4 kb
-1.9
kb
(W HindIll
EC&I
’ 7.2kb (mbio4 12)
<
L
M;~~f;rr;g$d
----+tl.gkb+-
HindIll
HindIll
z$.z$kb __)
in
Fig. 3. (A) The restriction patterns of the mutated plasmids. Plasmid DNAs for restriction endonuclease analysis were prepared from the 20 large-colony strains derived from a single small colony of SB412 (pLGM412) by alkali lysis procedures, and the EcoRI-Hind111 digest products were fractionated on 0.7% agarose gels. C represents reference plasmid, pLGM412. A indicates HindIII-digested A DNA (size markers). A 7.2-kb fragment corresponds to the fragment containing the bio operon. 4.4- and 1.9-kb fragments are derivatives of the vector plasmid, pLG339. Lanes 1, 5, 8, 13, 16. 17, 18, 19, and 20 represent identical-type derivatives. Lanes 3, 4, 6, 10, 12, 14, and 15 are deleted-type derivatives. Lanes 2, 7, Y, and 11 show elongated-type derivatives. (B) Restriction map of pLGM412 and the location of the inserted DNA fragment in elongated-type plasmids.
N. Sakurai et al. /Journal
of Biotechnology 43 (1995) 11-l 9
tion sites and the fragment sizes. These plasmids were possibly subject to a point mutation, a small deletion, or an insertion by a small fragment. 3.5. Characterization of the mutated plasmids iso-
lated from large-colony strains To confirm whether the altered size of the colonies might correlate to a specific genetic lesion, the three types of the mutated plasmids recovered from the large colonies were introduced into SB412. The resulting transformants were tested for the colony size and the d-biotin production. In addition, these mutated plasmids were introduced into E. coli Biostrains and the resultant recombinant strains were tested for complementation of the Bio- phenotypes (Table 2). All of the transformants with the above three types of the mutated plasmids produced large colonies. Subsequently, we tested these transformants for d-biotin production by using medium F2. The deleted-type plasmids showing BioB- (plasmid D-l as a representative) or BioA-B-F-C-D(plasmid D-2 as a representative) phenotype and SB412 with these plasmids produced d-biotin at about 35 mg 1- ‘. All of the elongated-type plasmids exhibited BioB- phenotype (plasmids E-l and E-2 d-Biotin productions of as representatives). SB412(E-1) and SB412(E-2) were 32 and 42 mg 1 - ‘, respectively. Interestingly, two-thirds of the identical-type plasmids exhibited BioB+ phenotype. These identical-type plasmids showing BioB+ (P-l as a representative) and BioB- (P-2 as a representative) phenotype introduced into SB412; resulting transformants formed large colonies and produced d-biotin at 110 mg 1-l and 36 mg l-‘, respectively. The d-biotin production of SB412(P-1) was similar to that of SB412(pLGWlOl), suggesting that the mutation in P-l would decrease the level of transcription and/or translation of the SB412 bioB gene to the level of the wild-type bioB genes. The results described here indicated that the bioB product might be significant for d-biotin overproduction in SB412.
17
which occurred in the large-colony strains tested included deletions or alterations of the bioB region. Quantitative measurement of biotin synthase, however, has not been reported. Thus, to confirm our assumption, we constructed pLG339 derivatives carrying the wild-type or SB412 bioB gene alone. Resultant plasmids, pLGWlOlB and pLGM412B, were introduced into SB412 and the colony sizes of the transformants were observed. The colony size of SB412(pLGM412B) was identical to that of SB412(pLGM412), whereas SB412(pLGWlOlB) produced normal-sized colonies, similar to SB412cpLG339). Large colonies appeared after incubation for more than 3 d from the background small-colonies of SB412(pLGM412B). This result suggested that the growth inhibition of SB412(pLGM412) would be caused from the highly expressed bioB product, biotin synthase, but might not be due to either the shortage of the precursors before dethiobiotin or high expressions of the other bio genes. Further, we observed that several largecolony strains with the identical-type plasmids showing BioB- phenotype produced similar amounts of inactive biotin synthase to that of normal SB412(pLGM412) cells (Fig. 4, lanes 4 and 5). This results suggested that the bioB protein itself might not be toxic to SB412 host. 3.7. Introduction of a recA mutation Although SB412(pLGM412) showed a very poor growth and structural mutations of the plasmid, this recombinant strain was expected to have a potential Ml2345678
3.6. Identification of the gene responsible for the
growth inhibition The results described above suggested that a high expression of the bioB gene would be responsible for the growth inhibition since all of the mutations
Fig. 4. SDS-PAGE analysis of cell extracts from large colonies. M indicates molecular markers. Lanes l-8 contain total cellular proteins from SB412 host carrying the following plasmids: 1, normal pLGM412; 2-5, identical-type derivatives from large-colonies; 6 and 7, deleted-type derivatives from large-colonies; 8, pLGM304.
IX
N. Sakurai et al. /Journal
for high d-biotin production. For example, optimizing the cultural conditions and searching for the growth promoting substances might help the increase of d-biotin production by SB412CpLGM412). Plasmid instability is a primary impediment to the industrial utilization of recombinant microorganisms. This instability, in general, is caused by the stress resulting from the metabolic burden because of the high expression of the plasmid-encoded products. The structural instability of the plasmid might be overcome by the introduction of the recA mutation since the structural instabilities are associated with DNA recombination by the action of RecA (Kate and Shinoura, 1977). In an attempt to minimize the instability by a RecA-mediated recombination, a recA mutation was introduced into SB412 by the chromosomal insertion of Mu dl phage. Several Ap’ strains showing ultraviolet-sensitive phenotype were obtained. Sequencing and computer analyses revealed that the recA genes were disrupted in the two Ap’ strains, SB412Rl and SB412R2, (unpublished data). Next, pLGM412 was introduced into these recA mutants. The resultant small colonies of SB412RlcpLGM412) and SB412R2(pLGM412) were tested for the plasmid stability by evaluating the frequency of large colonies generated from small. The recA mutation, however, did not affect the frequency of the structural instability. The large-colony type mutants were found and the frequency of the large colonies were similar to that of SB412(pLGM412) (9 X lo-‘; Sakurai et al., 1993b). 3.8. Conclusion We have demonstrated that highly expressed biotin synthase, the rate limiting enzyme for d-biotin synthesis (Sakurai et al., 1993b), causes strong growth inhibition. Although the reaction mechanism of biotin synthase has not been elucidated yet (Parry, 1983; Ifuku et al., 1992), the introduction of sulfur atom to dethiobiotin apparently proceeds via an oxidative reaction or a conjugated redox reaction since biotin is chemically an oxidative form of dethiobiotin. Thus, it is possible that oxidative coproducts such as peroxidants and/or radical species might be generated along with the biotin synthase reaction. These reactive co-products might be harmful to the intact cells, causing a growth inhibition.
of Biotechnology 43 (1995) 11-19 For the industrialization of the d-biotin process, we need to obtain mutants producing a larger amount of d-biotin than SB412(pLGM304), but the growth inhibition by biotin synthase makes it difficult to construct a genetically stable d-biotin-producing strain. Nevertheless, this difficulty can be overcome by finding the exact mechanism of this growth inhibition.
References Ball, T.K., Wasmuth, C.R., Braunagel, S.C. and Benedik, M.J. (1990) Expression of Serratia marcescens extracellular proteins requires recA. J. Bacterial. 172, 342-349. Boliver, F., Rodriguez, R.L., Greene, P.J., Betlach, H.L., Heynecker, H.W., Boyer, H.W., Crosa, J.H. and Falkow, S. (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95-113. Boyer, H.W. and Roulland-Dussoix, D. (1969) A complementation of the restriction and modification of DNA in Escherichia cob. J. Mol. Biol. 41, 459-472. Casadaban, M.J. and Cohen, S.N. (1979) Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc. Natl. Acad. Sci. USA 76, 4530-4533. Davis, B.D. and Mingioli, ES. (1950) Mutants of Escherichia coli requiring methionine or vitamin B,,. J. Bacterial. 60, 17-28. Del Campillo-Campbell, A., Kayajanian, G., Campbell, A. and Adhya, S. (1967) Biotin requiring mutants of Escherichia coli K-12. J. Bacterial. 94, 2065-2066. Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M. and Fukushima, Y. (1992) Conversion of dethiobiotin to biotin in cell-free extracts of Escherichia coli. Biosci. Biotechnol. Biochem. 56, 1780-1785. Kato, T. and Shinoura, Y. (1977) Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol. Gen. Genet. 156, 121-131. Maniatis, T., Fritsh, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. Matsumoto, H., Tazaki, T. and Hosogaya, S. (1973) A generalized transducing phage of Serratia marcescens. Jpn. J. Microbial. 17, 473-479. Parry, R.J. (1983) Biosynthesis of some sulfur containing natural products. Investigations of the mechanism of carbon-sulfur bond formation. Tetrahedron 39, 1212-1238. Sakurai, N., Imai, Y., Masuda, M., Komatsubara, S. and Tosa, T. (1993a) Construction of a biotin-overproducing strain of Serratia marcescens. Appl. Environ. Microbial. 2857-2863. Sakurai, N., Imai, Y., Masuda, M., Komatsubara, S. and Tosa, T. (1993b) Molecular breeding of a biotin-hyperproducing Serratia marcexerts strain. Appl. Environ. Microbial. 59, 32253232.
N. Sakurai et al. /Journal Snell, E.E., Eakin, R.E. and Williams, R.J. (1940) A quantitative test for biotin and observations regarding its occurrence and properties. J. Biol. Chem. 62, 175-178. Stoker, N.G., Fairweather, N.F. and Spratt, B.G. (1982) Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene 18, 335-341.
of Biotechnology
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19
Takagi, T. and Kisumi, M. (1985) Isolation of a versatile Serratia marcescens mutant as a host and molecular cloning of the aspartase gene. J. Bacterial. 161, l-6. Van de Patte, P., Cramer, S. and Giphart-Gassier, M. (1980) Invertible DNA determines host specificity of bacteriophage Mu. Nature (Iond.) 286, 218-222.
43 (1995) 11-19
Instability of the mutated biotin operon plasmid in a biotin-producing mutant of Serratia marcescens Naoki Sakurai apbT *, Yuji Imai a, Saburo Komatsubara
ayb
a Research Laboratory ofApplied Biochemistry, Tanabe Seiyaku Co., Ltd., 16-89 Kashima-3-chome, Yodogawa-ku, Osaka 532, Japan b Research Laboratory ofApplied
Biochemistry
at Toda, Tanabe Seiyaku Co., Ltd., 2-50, Kawagishi-2-chome, Japan
Received 8 September
Toda-shi, Saitama 335,
1994; revised 24 April 1995; accepted 22 May 1995
Abstract The growth of a d-biotin-producing strain of Serratia marcescens (SB412) was strongly inhibited by the introduction of pLGM412, a low-copy-number plasmid containing the complete biotin (bio) operon derived from SB412, whereas the wild-type strain was not inhibited by the plasmid. SB412 carrying pLGM412 was genetically unstable; large colonies appeared spontaneously from the background small colonies. When the plasmids from the large colonies were transformed into the SB412 host, all of the resultant transformants showed a large-colony phenotype, suggesting that the large-colony phenotype is due to mutations in the plasmid-born bio genes. Some of these plasmids were structurally altered and the others
were not. Furthermore, the structurally altered plasmids were classified into a deleted and an elongated type. All of the mutated pLGM412 derivatives reduced or lacked the bio gene expression, indicating that the high expression of bio gene(s) causes the growth inhibition. growth inhibition. Keywords:
d-Biotin producing
By subcloning
experiments,
strain; Serratia marcescens;
biotin synthase (the bioB gene product) was responsible
Growth inhibition;
1. Introduction We have been studying d-biotin production by regulatory mutants of Serratia marcescens Sr41 and have shown alterations in d-biotin-mediated feedback repression of biotin biosynthesis in these mutants (Sakurai et al., 1993a). Strain SB304, resistant to a lower concentration of a biotin analog, acidomycin, produced d-biotin at 5 mg 1-l and strain SB412, derived from SB304 and resistant to a
* Corresponding author: Research Laboratory of Applied Biochemistry at Toda, Tanabe Seiyaku Co., Ltd., 2-50, Kawagishi-2chome, Toda-shi, Saitama 335, Japan. 0168-1656/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1656(95)00103-4
Large colony; Mutated
for the
bio gene; Biotin synthase
higher concentration of acidomycin, produced d-biotin at 20 mg 1-l (Sakurai et al., 1993a). Genetic analyses have revealed that the primary and secondary mutations for d-biotin overproduction occurred in the bio genes of SB304 and SB412, respectively. Subsequently, we cloned the mutated bio operon and found that the two strains with the cloned bio operon showed a high d-biotin production (Sakurai et al., 1993b). Strain SB412 harboring plasa low-copy-number plasmid mid pLGM304, (pLG339) containing the mutated bio operon of SB304, produced d-biotin at 200 mg 1-l in a shaking flask. Since SB412 had two mutations in the bio genes
.V. Sakurut et ~1. /.lountul
12
of Biotechnology
cluster for d-biotin overproduction, we expected that the most productive strain would be obtained by transforming SB412 with pLGM412, a pLG339 derivative containing the SB412 hio operon or by the gene dosage effect of the ho genes in a highcopy-number plasmid. However. we failed to obtain such strains because of a strong growth inhibition by these recombinant plasmids. Here we report the growth inhibition of a biotinproducing strain by the mutated hio genes in plasmid.
43 (1995)
I I-l 9
0.5% glucose, 1.0% peptone, 0.3% meat extract, 1.0% yeast extract, and 0.5% NaCl was used for routine colony isolation and growth study. The minimal medium of Davis and Mingioli (1950) was modified by omitting sodium citrate and increasing the glucose concentration to 0.5%. Medium F2, used for d-biotin production, contained 15% sucrose, 1.5% urea, 0.1% corn steep liquor, 0.1% K,HPO,, 0.2% MgSO, .7H,O, 0.01% FeSO, .7H,O, and 1.5% CaCO,. 2.2. d-Biotin production
2. Materials
and methods
2.1. Strains, plasmids,
and media
Bacterial strains and plasmids used in this work are listed in Table I. Nutrient medium containing
Cells were cultured in a shaking flask containing medium F2 as described previously (Sakurai et al.. 1993b). d-Biotin was assayed by the method of Snell et al. (I 940). Biotin-related compounds, which correspond to d-biotin plus dethiobiotin, were assayed as described previously (Sakurai et al., 1993a).
Table I Bacterial strains and plasmids used Strain and plasmid
Characteristic
E. co/r strains HBIOI
”
Ref.
Fan hsaS-70 (I~. mn)
resA13 uru-14proA2
rhi-I lucYI galK2 rpsL20 xyl-5 mtl-I supE44
MALI01 R879 R87S R872 R87h RX77 S. IIIU~C~~~II.Sstrains 8000 TT3’): SB412 SB412RI SB412R2 Plasmids pLG339 pBR327 pBWlO1 pBM412 pLGWlO1 pLGM304 pLGM412 pLGWlOlB pLGM412B
F
J
bid-74 hioBl7 hioF
( gpt-proAB-argF-luc) IN (rmD-rmE) IN ( rmD-rrE)
I
IN (rmD-rrnE)
I
IN CrrnD-r(nE:’
wild type Nut r resistant to 2.0 mg of acidomycin rrtA::Mu dl rccA::Mu dl Km’ Tc’ pur AP’ pBR322::wbio Ap’ pBR322::mbiu,,, Ap’ pLG33Y::wbio Km’ pLG33Y::mbiu,,, Km’ pLG339::mbro,,, Km’ pLG339::wbioB Km’
-
pLG339::mbioB,,,
Km’
I
I
per ml
Bayer and Roulland-Dussoix,
A
XIII rpsL [Mu dl (lx,
I
hioC’IX (~hlAlX IN (rrrtD-rrnE1 hroDl9
leuB6
AP)]
used for plasmid constructions Casadaban and Cohen, 1979 Del Campillo-Campbell et al., Del Campillo-Campbell et al., Del Campillo-Campbell et al., Del Campillo-Campbell et al., Del Campillo-Campbell et al.,
1969;
1967 1967 1967 1967 1967
Matsumoto et al.. 1973 Takagi and Kisumi, 1985 Sakurai et al., 1993a this study (mutant from strain SB412) this study (mutant from strain SB412) Stoker et al., 1982 Bolivcr et al., 1977 Sakurai et al., 1993b Sakurai et al., 1993b Sakurai et al., 1993b Sakurai et al., 1993b Sakurai et al., 1993b this study this study
’ Symbols used for relevant genotypes and phenotypes arc as follows: Nut-, no production of extracellular nuclease; I-, defect of host restriction enzyme; pur. active plasmid partitioning; whio, wild-type biotin operon; mbiojo,, biotin operon allele of SB304; mbio,tz, biotin operon allele of SB412; wbioB. wild-type hi& gene; mbioBj,r. bioB gene of SB412.
N. Sakurai et al./Journal
2.3. Genetic procedures niques
and recombinant
ofBiotechnology 43 (1995) 11-19
of the cell suspension (lo4 to lo5 cells per ml) was spread on nutrient agar plates. After 3 d of incubation at 30°C large colonies found among small colonies were scored. Subsequently, plasmids of large colonies were analyzed.
DNA tech-
Standard procedures were used for plasmid preparation, restriction enzyme digestion, ligation, and agarose gel electrophoresis (Maniatis et al., 1982). Transformation of Escherichia coli and S. marcescens was carried out according Maniatis et al. (1982). 2.4. Analysis
of plasmid
2.5. Curing of the plasmids from large-colony-forming derioatives of SB412cpLGM412) Cells of large-colony-forming derivatives of SB412(pLGM412) were cultured in 3 ml of nutrient medium for 48 h at 30°C with shaking. Cells were diluted with saline and spread onto nutrient agar plates without kanamycin. Colonies formed were tested for the kanamycin sensitivity and the presence of plasmids.
instability
A single small colony of SB412(pLGM412) grown on a nutrient agar plate was inoculated into a test tube containing 3 ml of a nutrient medium. Incubation was continued at 30°C for 24 h with shaking. The culture was diluted with saline. A O.l-ml aliquot Hidll
ECdl
f-------7.2
13
HindIll
kb ____jt
pBM412 (or pBW101) Aval-Stuldigestion
I
Hincll
digestion
HindIll
Fig. 1. Restriction
maps of pLGWlOlB
and pLGM412B
containing
the bioB
genes of the wild strain and SB412, respectively.
IV. Sakurur et al./Journul
I4
ofBiotechnology 43
(1995) II-19
2.6. Plasmid constructions Plasmids pLGW101B and pLGM412B were constructed as follows. The 2.0-kb Al,aI-StuI fragments carrying the bioB gene and its promoter region were excised from pBWlO1 and pBM412, and were ligated into the HiilcII site of pLG339, producing pLGWlOlB and pLGM412B, respectively (Fig. I ). 2.7. Construction
of the recA mutant
Mu dl phage was used to isolate the recA mutant of SB412. Mu dl is a derivative of phage Mu which contains an ampicillin-resistance marker and can insert its DNA into the chromosomes of various bacteria (Van de Patte et al.. 1980). Lysate obtained by heat induction of E. co/i MAL103 was used to create random Mu dl chromosomal insertions in ampicillin-resistant colonies were SB412. First, screened for the recA mutation by measuring their sensitivity to ultraviolet irradiation. Further, the locus destructed by Mu dl were cloned from the candidate mutants by using ampicillin resistance carried by Mu dl as the selective marker. The recA mutations were confirmed by sequencing the cloned genes, and the sequences were compared with that of the S. marcescens recA gene reported by Ball et al. ( 1990). 2.X Chemicals d-Biotin was a product of Tanabe Seiyaku C’o.. Ltd. Dethiobiotin was prepared and given by Production Technology Division of Tanabe Seiyaku Co.. Ltd. Restriction endonucleases and T4 DNA ligase were purchased from Takara Shuzo Co., Ltd., Kyoto, Japan. Other chemicals were also obtained commercially and not purified further.
3. Results and discussion 3.1. Growth inhibition operon plasmid
ofSB412
by the mutated bio
The mutations for d-biotin overproduction occurred in the bio genes of strain SB412 (Sakurai et al., 1993a). To construct a more productive strain,
Fig. 2. Colony sizes of strains 8000 (wild type) and SB412 Cd-biotin-producing mutant) carrying recombinant plasmids of the hio operons. Cells obtained by single colony isolation were grown in a nutrient medium containing kanamycin for 24 h and spread onto nutrient agar plates containing kanamycin. The plates were incubated for 2 d at 30°C except the plates for SB412(pLGM412), which was incubated for 3 d at 30°C. Photographs: (A). 8000(pLGM412); (B), XOOtKpLGM304); CC), 8000(pLG33Y); CD). SB412cpLGM412); (E). SB412(pLGM304); (F), SB412(pLG33Y).
SB412 was transformed with high-copy-number plasmids, pBM304 and pBM412, consisted of a pBR322 replicon and the mutated bio genes of SB304 and SB412, respectively. These two plasmids, however, could not be introduced into SB412 host under various transformation conditions, whereas strain 8000, wild-type of S. marcescens. was transformed with them at high frequencies. Then, WC attempted to introduce pLGM412, a low-copynumber plasmid with SB412 bio genes into SB412. Transformants of SB412 bearing pLGM412 yielded a few large colonies but the majority of colonies were barely visible after incubation for 3 d at 30°C on nutrient plates containing kanamycin (Fig. 3). These small colonies exhibited very poor growth when transferred to fresh kanamycin nutrient plates and grew to very low densities even when incubated for 2 to 3 d in liquid kanamycin nutrient medium. However, all of the transformants showing large-colony phenotype grew fast as did SB412 (Fig. 2). The colonies were uniform in size and similar in size to that of SB412, a host strain, on kanamycin nutrient plates, and grew to high density in kanamycin nutrient liquid medium. Under the conditions, SB412 carrying pLG339 and pLGW101, a pLG339 deriva-
N. Sakurai et al. /Journal
of Biorechnology 43 (1995) 11 -I 9
tive with the wild-type bio genes, exhibited the similar colonies in size. In contrast, the introduction of pLGM412 into strain 8000 (wild strain) did not cause growth inhibition. Thus, the growth inhibition by the introduction of pLGM412 occurred in SB412 background, suggesting that the growth inhibition by pLGM412 might be due to the high expression of the bio gene(s). Under the conditions described above, no large colonies were generated from SB412 carrying pLGM304, although the colony size of this strain was half that of SB412. To examine the profile of the growth inhibition observed in SB412(pLGM412), we analyzed the host physiology and the plasmids from large colonies generated from small colonies.
SB412(pLGWlOl) (Table 21, indicating that mutations for reducing d-biotin production occurred in either host cells or plasmids in the large-colony strains.
3.3. Curing of plasmids from large-colony strains To determine whether the mutation for decreased d-biotin production in large-colony strains resided in the host strain or in the plasmid, plasmids of ten large-colony strains isolated independently were cured of the plasmids and resulting cured strains were tested for d-biotin production. These cured strains accumulated d-biotin at a concentration of about 40 mg 1-l as did SB412. This result suggested that no genetic alterations for the reduction of d-biotin production occurred in the host cells of large-colony strains. Subsequently, we re-transformed the cells of SB412 with plasmid DNAs extracted from each of above ten large-colony strains. d-Biotin production of resulting recombinants was between 40 and 110 mg 1 - ’ (Table 2).
3.2. d-Biotin production by large-colony strains First, we examined d-biotin production by 30 large-colony strains obtained from independent small colonies of SB412(pLGM412). The amounts of dbiotin produced ranged from 40 mg 1-l to 110 mg 1-l. The former value was identical to that of SB412 and the latter value was similar to that of
Table 2 Characteristics
of pLGM412
Derivatives of pLGM412 (type of mutation) a
pLG339 (vector) pLGW101 pLGM304 D-l (deleted) D-2 (deleted) E-l (elongated) E;2 (elongated) P-l (identical) P-2 (identical)
15
derivatives Complementation bioA
bioB
b
Productivity bioF
bioC
_
_e +’ + + + + + +
bioD
1 + _ _ _ + _
1 + + _
1 + + _
+ + +
+ + + +
+ + + +
+ + + +
of SB412 transformants
d-Biotin
Biotin-related
(mg 1-l 1
(mg 1-t)
35 100 190 36 33 32 42 110 36
50 120 310 52 45 48 58 200 53
’
compounds
d
a Derivatives of pLGM412 were extracted from the large colonies of SB412 (pLGM412) and the restriction patterns were examined with 0.7% agarose-gel electrophoresis. b The plasmid DNAs were introduced into E. coli biotin auxotrophs (R879, R875, R872, R876, R877) and the resultant transformants were tested for complementation of Bio- phenotype. ’ The mutant plasmids which are representatives of each type were introduced into SB412. Cells of the re-transformants were grown at 30°C for 120 h in shaking flasks containing medium F2. A reference strain, SB412, produced 40 mg of d-biotin per liter under these conditions. d Total amount of d-biotin and dethiobiotin. e -, no growth. f +, growth.
N. Sakurai et d/Journal
16
3.4. Structural
changes
ofBiotechnology
of the plasmids of large-col-
ony strains
We analyzed restriction maps of the plasmid DNAs extracted from the each of 300 large-colony strains, which were derived from ten independent small colonies, as follows. Plasmid DNAs were prepared from the large-colony strains and were examined by size analysis on agarose gels (Fig. 3A). When pLGM412 was digested with EcoRI and HindIII, 1.9-, 4.4-, and 7.2-kb DNA bands were observed (Fig. 3B). 1.9- and 4.4-kb bands were derived from the vector plasmid, pLG339, and a 7.2-kb fragment contained the bio gene cluster. Fig. 3 shows a typical restriction pattern for 20 plasmids isolated from the large-colony strains derived from a small colony. The 7.2-kb fragment of seven plasmids (lanes 3, 4, 6, 10, 12, 14, and 15; deleted type) were
09
h 12
J3 (1995) 11-19
subject to deletion, in four plasmids (lanes 2, 7, 9, and 11; elongated type) were inserted by a short fragment of about 1 kb, and the rest (lanes 1, 5, 8, 13, 16, 17, 18, 19, and 20; identical type) had no detectable changes in the 7.2-kb fragment. The ratio of three types of the mutant plasmids in the 300 large colonies was 17% for deleted type, 74% for identical type, and 9% for elongated type. All of the deleted-type plasmids were found to lack either the full-length bio operon, a partial region of the bioB gene, or an entire region of the bioB gene. All of the elongated-type plasmids were inserted by an 1-kb additional fragments into the bioB structural gene. The site of the insertion was apparently the same in the independent elongated types as judged by restriction analysis. The origin of the inserted DNA, however, has not been specified yet. Identical-type plasmids were very similar to pLGM412 in the restric-
3 4 5 6 7 8 9ltlIlPl314l5l617BlPzIC
-7.2 -
kb 4.4 kb
-1.9
kb
(W HindIll
EC&I
’ 7.2kb (mbio4 12)
<
L
M;~~f;rr;g$d
----+tl.gkb+-
HindIll
HindIll
z$.z$kb __)
in
Fig. 3. (A) The restriction patterns of the mutated plasmids. Plasmid DNAs for restriction endonuclease analysis were prepared from the 20 large-colony strains derived from a single small colony of SB412 (pLGM412) by alkali lysis procedures, and the EcoRI-Hind111 digest products were fractionated on 0.7% agarose gels. C represents reference plasmid, pLGM412. A indicates HindIII-digested A DNA (size markers). A 7.2-kb fragment corresponds to the fragment containing the bio operon. 4.4- and 1.9-kb fragments are derivatives of the vector plasmid, pLG339. Lanes 1, 5, 8, 13, 16. 17, 18, 19, and 20 represent identical-type derivatives. Lanes 3, 4, 6, 10, 12, 14, and 15 are deleted-type derivatives. Lanes 2, 7, Y, and 11 show elongated-type derivatives. (B) Restriction map of pLGM412 and the location of the inserted DNA fragment in elongated-type plasmids.
N. Sakurai et al. /Journal
of Biotechnology 43 (1995) 11-l 9
tion sites and the fragment sizes. These plasmids were possibly subject to a point mutation, a small deletion, or an insertion by a small fragment. 3.5. Characterization of the mutated plasmids iso-
lated from large-colony strains To confirm whether the altered size of the colonies might correlate to a specific genetic lesion, the three types of the mutated plasmids recovered from the large colonies were introduced into SB412. The resulting transformants were tested for the colony size and the d-biotin production. In addition, these mutated plasmids were introduced into E. coli Biostrains and the resultant recombinant strains were tested for complementation of the Bio- phenotypes (Table 2). All of the transformants with the above three types of the mutated plasmids produced large colonies. Subsequently, we tested these transformants for d-biotin production by using medium F2. The deleted-type plasmids showing BioB- (plasmid D-l as a representative) or BioA-B-F-C-D(plasmid D-2 as a representative) phenotype and SB412 with these plasmids produced d-biotin at about 35 mg 1- ‘. All of the elongated-type plasmids exhibited BioB- phenotype (plasmids E-l and E-2 d-Biotin productions of as representatives). SB412(E-1) and SB412(E-2) were 32 and 42 mg 1 - ‘, respectively. Interestingly, two-thirds of the identical-type plasmids exhibited BioB+ phenotype. These identical-type plasmids showing BioB+ (P-l as a representative) and BioB- (P-2 as a representative) phenotype introduced into SB412; resulting transformants formed large colonies and produced d-biotin at 110 mg 1-l and 36 mg l-‘, respectively. The d-biotin production of SB412(P-1) was similar to that of SB412(pLGWlOl), suggesting that the mutation in P-l would decrease the level of transcription and/or translation of the SB412 bioB gene to the level of the wild-type bioB genes. The results described here indicated that the bioB product might be significant for d-biotin overproduction in SB412.
17
which occurred in the large-colony strains tested included deletions or alterations of the bioB region. Quantitative measurement of biotin synthase, however, has not been reported. Thus, to confirm our assumption, we constructed pLG339 derivatives carrying the wild-type or SB412 bioB gene alone. Resultant plasmids, pLGWlOlB and pLGM412B, were introduced into SB412 and the colony sizes of the transformants were observed. The colony size of SB412(pLGM412B) was identical to that of SB412(pLGM412), whereas SB412(pLGWlOlB) produced normal-sized colonies, similar to SB412cpLG339). Large colonies appeared after incubation for more than 3 d from the background small-colonies of SB412(pLGM412B). This result suggested that the growth inhibition of SB412(pLGM412) would be caused from the highly expressed bioB product, biotin synthase, but might not be due to either the shortage of the precursors before dethiobiotin or high expressions of the other bio genes. Further, we observed that several largecolony strains with the identical-type plasmids showing BioB- phenotype produced similar amounts of inactive biotin synthase to that of normal SB412(pLGM412) cells (Fig. 4, lanes 4 and 5). This results suggested that the bioB protein itself might not be toxic to SB412 host. 3.7. Introduction of a recA mutation Although SB412(pLGM412) showed a very poor growth and structural mutations of the plasmid, this recombinant strain was expected to have a potential Ml2345678
3.6. Identification of the gene responsible for the
growth inhibition The results described above suggested that a high expression of the bioB gene would be responsible for the growth inhibition since all of the mutations
Fig. 4. SDS-PAGE analysis of cell extracts from large colonies. M indicates molecular markers. Lanes l-8 contain total cellular proteins from SB412 host carrying the following plasmids: 1, normal pLGM412; 2-5, identical-type derivatives from large-colonies; 6 and 7, deleted-type derivatives from large-colonies; 8, pLGM304.
IX
N. Sakurai et al. /Journal
for high d-biotin production. For example, optimizing the cultural conditions and searching for the growth promoting substances might help the increase of d-biotin production by SB412CpLGM412). Plasmid instability is a primary impediment to the industrial utilization of recombinant microorganisms. This instability, in general, is caused by the stress resulting from the metabolic burden because of the high expression of the plasmid-encoded products. The structural instability of the plasmid might be overcome by the introduction of the recA mutation since the structural instabilities are associated with DNA recombination by the action of RecA (Kate and Shinoura, 1977). In an attempt to minimize the instability by a RecA-mediated recombination, a recA mutation was introduced into SB412 by the chromosomal insertion of Mu dl phage. Several Ap’ strains showing ultraviolet-sensitive phenotype were obtained. Sequencing and computer analyses revealed that the recA genes were disrupted in the two Ap’ strains, SB412Rl and SB412R2, (unpublished data). Next, pLGM412 was introduced into these recA mutants. The resultant small colonies of SB412RlcpLGM412) and SB412R2(pLGM412) were tested for the plasmid stability by evaluating the frequency of large colonies generated from small. The recA mutation, however, did not affect the frequency of the structural instability. The large-colony type mutants were found and the frequency of the large colonies were similar to that of SB412(pLGM412) (9 X lo-‘; Sakurai et al., 1993b). 3.8. Conclusion We have demonstrated that highly expressed biotin synthase, the rate limiting enzyme for d-biotin synthesis (Sakurai et al., 1993b), causes strong growth inhibition. Although the reaction mechanism of biotin synthase has not been elucidated yet (Parry, 1983; Ifuku et al., 1992), the introduction of sulfur atom to dethiobiotin apparently proceeds via an oxidative reaction or a conjugated redox reaction since biotin is chemically an oxidative form of dethiobiotin. Thus, it is possible that oxidative coproducts such as peroxidants and/or radical species might be generated along with the biotin synthase reaction. These reactive co-products might be harmful to the intact cells, causing a growth inhibition.
of Biotechnology 43 (1995) 11-19 For the industrialization of the d-biotin process, we need to obtain mutants producing a larger amount of d-biotin than SB412(pLGM304), but the growth inhibition by biotin synthase makes it difficult to construct a genetically stable d-biotin-producing strain. Nevertheless, this difficulty can be overcome by finding the exact mechanism of this growth inhibition.
References Ball, T.K., Wasmuth, C.R., Braunagel, S.C. and Benedik, M.J. (1990) Expression of Serratia marcescens extracellular proteins requires recA. J. Bacterial. 172, 342-349. Boliver, F., Rodriguez, R.L., Greene, P.J., Betlach, H.L., Heynecker, H.W., Boyer, H.W., Crosa, J.H. and Falkow, S. (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95-113. Boyer, H.W. and Roulland-Dussoix, D. (1969) A complementation of the restriction and modification of DNA in Escherichia cob. J. Mol. Biol. 41, 459-472. Casadaban, M.J. and Cohen, S.N. (1979) Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc. Natl. Acad. Sci. USA 76, 4530-4533. Davis, B.D. and Mingioli, ES. (1950) Mutants of Escherichia coli requiring methionine or vitamin B,,. J. Bacterial. 60, 17-28. Del Campillo-Campbell, A., Kayajanian, G., Campbell, A. and Adhya, S. (1967) Biotin requiring mutants of Escherichia coli K-12. J. Bacterial. 94, 2065-2066. Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M. and Fukushima, Y. (1992) Conversion of dethiobiotin to biotin in cell-free extracts of Escherichia coli. Biosci. Biotechnol. Biochem. 56, 1780-1785. Kato, T. and Shinoura, Y. (1977) Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol. Gen. Genet. 156, 121-131. Maniatis, T., Fritsh, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. Matsumoto, H., Tazaki, T. and Hosogaya, S. (1973) A generalized transducing phage of Serratia marcescens. Jpn. J. Microbial. 17, 473-479. Parry, R.J. (1983) Biosynthesis of some sulfur containing natural products. Investigations of the mechanism of carbon-sulfur bond formation. Tetrahedron 39, 1212-1238. Sakurai, N., Imai, Y., Masuda, M., Komatsubara, S. and Tosa, T. (1993a) Construction of a biotin-overproducing strain of Serratia marcescens. Appl. Environ. Microbial. 2857-2863. Sakurai, N., Imai, Y., Masuda, M., Komatsubara, S. and Tosa, T. (1993b) Molecular breeding of a biotin-hyperproducing Serratia marcexerts strain. Appl. Environ. Microbial. 59, 32253232.
N. Sakurai et al. /Journal Snell, E.E., Eakin, R.E. and Williams, R.J. (1940) A quantitative test for biotin and observations regarding its occurrence and properties. J. Biol. Chem. 62, 175-178. Stoker, N.G., Fairweather, N.F. and Spratt, B.G. (1982) Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene 18, 335-341.
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Takagi, T. and Kisumi, M. (1985) Isolation of a versatile Serratia marcescens mutant as a host and molecular cloning of the aspartase gene. J. Bacterial. 161, l-6. Van de Patte, P., Cramer, S. and Giphart-Gassier, M. (1980) Invertible DNA determines host specificity of bacteriophage Mu. Nature (Iond.) 286, 218-222.