Beruflich Dokumente
Kultur Dokumente
Received: 6 March 2002 / Revised: 3 October 2002 / Accepted: 11 October 2002 ' Published online: 9 November 2002
9 Springer-Verlag 2002
Table 1 Quantitative estimation of PHB production using gas chromatography. Values are expressed in terms of mean PHB content
(% dry weight)_+SD. N.D. Not determined
18 24 42 66 90 114
Streptomyces albus ATCC 21132 1.48 3.13 4.59 2.92 1.04 N.D.
+0.01 +0.0 l +0.02 _+0.01 +0.01
Streptomyces venezuelae ATCC 21113 3.49 1.71 1.02 0.12 N.D. N.D.
_+0.01 +0.01 +0.02 -+0.01
Streptomyces lividans ATCC 19844 1.27 2.07 1.87 0.83 0.41 0. I0
_+0.01 +0.02 +0.02 +0.02 __+0.02 _+0.01
Srreptomyces coelicolor MTCC (India) 8 2.16 3.39 2.17 0.69 0.40 N.D.
9+0.01 +0.01 +0.01 +0.02 +0.02
Streptomyces olivaceus MTCC (India) 1392 0.25 4.10 7.79 6.15 4.95 2.18
-+0.02 +0.02 _+0.02 __+0.02 _+0.02 _+0.01
Streptomyces sp. (University of Delhi, India) 1.49 3.13 4.59 2.92 1.04 N.D.
9+0.01 _+0.0 l _+0.03 +0.02 _+0.01
Streptomyces sp. ATCC 21175 1.59 1.46 0.55 0.24 N.D. N.D.
_+0.02 _+0.0l +0.01 _+0.01
Streptomycesfi'adiae ATCC 10745 1.86 2.67 3.33 6.4l 9.89 6.76
_+0.01 9+0.01 _+0.01 _+0.01 _+0.01 _+0.0I
Streptomyces rosa ATCC 27462 1.50 0.86 0.16 0.11 N.D. N.D.
9+0.01 9+0.01 +0.01 +0.01
Streptomycespwwus ATCC 12433 5.89 2.14 1.28 1.16 0.24 N.D.
9+0.01 9+0.01 -+0.02 +0.02 -+0.01
Streptomyces kanamyceticus ATCC 12853 1.54 0.43 0.27 N.D. N.D. N.D.
9+0.01 9+0.02 +0.02
Streptomyces coelicolor A3(2) M145 (University of Cambridge, UK) 3.62 7.78 9.76 11.8 8.40 7.23
_+0.01 +0.01 -+0.0 l -+0.02 -+0.02 -+0.01
68
I00 ,2 a ~1.6
,72
90
8O
70
t~2 E
%T 60
- 1.o
50
40 -08
30
- 06 i~
20
10 - O.4
0 I / I I I I
~2
4000 3200 2400 2000 1600 1200 800 400 g
100
o' . . . . . . . . 0.0
%T
60
50
b - 1.6 120
40 1 7 2 Y
- 14
too
30
- 12
20
- 80
10 - 1.0
0 ._e
| | i i i |
-0.8
4000 3200 2400 2000 1600 1200 800 400 - 60 i
IO0
- 0.6
- 40
9o C
80 - 0.4
70 " X . . / - , , # - 0.2
- 20
%T 60
50 0.0 -0
40 20 40 60 80 100 120 140 160 180 200
30 Time (h)
1728Y
20
10 Fig. 2 a The temporal variation of PHB accumulation, and changes
0 in the pH and glucose concentration of the medium during growth
i
4000 3200
i
2400 2000
i m
1600 12GO
N i
800 400
of Streptomyces coelicolor A3(2) M145. 21 PHB, 0 pH, 9 glu-
cose, 9 dry cell weight, b The temporal variation of PHB accu-
W a v e n u m b e r ( c m "1) mulation and antibiotic production during growth of Streptomyces
coelicolor A3(2) M145. ~1 PHB, @ actinorhodin, 9 y-actino-
Fig. 1 Fourier transform-infrared absorption spectra of mycelial rhodin, A undecylprodigiosin
mass (a), of isolated poly-3-hydroxybutyric acid (PHB) (b) of
Streptomyces coelicolor A3(2) M145, and standard PHB from
Sigma (St.Louis, Mo., USA) (e)
color A3(2) M145 mainly produces three kinds of antibi-
otics, actinorhodin, ?-actinorhodin, and undecylprodigio-
imposable, definitively confirming the presence of PHB sin. The concentration of these three antibiotics was found
in the cells of S. coelicolor A3(2) M145. to increase gradually in the stationary phase of growth of
Since the role(s) of PHB in Streptomyces is not well the organism. Interestingly, there was a rapid increase in
understood, the possibility of a correlation between an- the concentration of actinorhodin and ?-actinorhodin coin-
tibiotic production and PHB utilization in S. coelicolor cident with the decrease in PHB concentration (Fig.2b).
A3(2) M145 was investigated. The strain was grown in This strongly indicated a possible role of the polymer in
SMM media for 8 days, and the temporal variation of a antibiotic production, probably as a source of the precur-
large number of parameters was measured (Fig. 2). The sor acetyl-CoA. A similar role has been found in S. vene-
organism reached the stationary phase of growth after zuelae and S. hygroscopicus. As the PHB concentration
3 days, the pH of the medium decreased from 5.5 to 4.4, decreased further, the concentration of undecylprodi-
and the glucose concentration in the medium decreased giosin also began to increase and continued increasing un-
rapidly, from 10 to 4.25 g/l, followed by a gradual de- til 192 h of growth (Fig. 2b). Primary metabolites provide
crease from 4.25 to 3.58 g/1. In the initial rapid phase of building blocks for the production of secondary metabo-
glucose consumption, PHB was produced, which indi- lites in the stationary phase of growth, when the organism
cates the utilization of glucose for growth and production stops growing and the accumulation of primary metabo-
of primary metabolites including PHB. lites could be potentially harmful. However, further ex-
PHB production increased rapidly during the exponen- periments involving labeling studies or PHB-negative
tial growth phase of the organism, with maximal produc- mutants of S. coelicolor A3(2) M145 are required to ab-
tion of 10.8% dry cell weight after 71 h of growth, after solutely confirm the role of PHB.
which the PHB concentration decreased rapidly (Fig. 2). In conclusion, all Streptomyces strains investigated in
This observation indicated utilization of the polymer by this study produced PHB, albeit in not very large quanti-
the cells during the stationary phase of growth. S. coeli- ties. In S. coelicolor A3(2) M145, the polymer may play a
69
rote in antibiotic biosynthesis, possibly by p r o v i d i n g pre- Kannan LV, Rehacek Z (1970) Formation of poly-~3-hydroxybu-
cursor m o l e c u l e s in the form o f a c e t y l - C o A . tyrate by actinomycetes. [nd J Biochem 7:126-129
Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000)
Acknowledgements We thank Prof. P. F. Leadlay, University of Practical Streptornyces genetics, The John Innes Foundation.
Cambridge, UK for kindly proving S. coelicolor A3(2) M145. S. Norwich
Verma was provided financial support by the I.I.T., Delhi, INDIA. Law JH, Slepecky RA (1961) Assay of poly-[3-hydroxybutyric
S.P.Valappil is being provided financial support by the University acid. J Bacteriol 82:33-36
of Westminster, London, UK. Finally, we thank Professor Chris Manna A, Banerjee R, Paul AK (1999) Accumulation of poly (3-
Bucke, University of Westminster, London. UK, for his constant hydroxybutyric acid) by some soil Streptomyces. Curt Micro-
encouragement and comments on the manuscript. bio! 39:153-158
Packter NM, Flatman S (1983) Characterization of acetoacetyl-
CoA reductase (3-oxoreductase) from Streptomyces coelicolor:
its possible role in polyhydroxybutyrate biosynthesis. Biochem
References Soc Trans 11:598-599
Ranade N, Vining LC (1993) Accumulation of intracellular carbon
Braunegg G, Sonnleitner B, Lafferty RM (1978) A rapid gas chro- reserves in relation to chloramphenicol biosynthesis by Strep-
matographic method for the determination of poly-~3-hydroxy- tomyces venezuelae. Can J Microbiol 39:377-383
butyric acid in microbial biomass. Eur J Appl Microbiol Bio- Steinbuchel A (1996) PHB and other polyhydroxyalkanoic.acids.
technol 6:29-37 In Rehm HJ, Reed G, Publer A Stadler P (eds) Products of pri-
Bystrykh LV, Fernandez-Moreno MA, Herrema JK, Malpartida F, mary metabolism. Biotechnology, vol 6. VCH, Weinheim
Hopwood DA and Dijkhuizen L (1996) Production of acti- Tsao SW, Rudd BAM, He X, Chang C, Floss HG (1985) Identifi-
norhodin-related 'blue pigments' by Streptornyces coelicolor cation of a red pigment from Streptomyces coelicolor A3(2) as
A3 (2). J Bacteriol 178:2238-2244 a mixture of prodigiosin derivatives. J Antibiotics 38:128-131
Hong K, Sun S, Tian W, Chen GQ, Huang W (1999) A rapid Wu K, Chang L, Revill PW, Katz L, Reeves CD (2000) The
method for detecting bacterial polyhyroxyalkanoates in intact FK520 gene cluster of Streptomyces hygroscopicus var. as-
cells by Fourier transform infrared spectroscopy. Appl Micro- comyceticus (ATCC 14891) contains genes for biosynthesis of
biol Biotechnol 51:523-526 unusual polykettide extender units. Gene 251:81-90