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Arch Microbiol (2002) 179:66~9

DOI 10. I007/s00203-002-0500-9

Shivani Verma 9 Yukti Bhatia


Sabeel Padinhara Valappil 9 Ipsita Roy

A possible role of poly-3-hydroxybutyric acid


in antibiotic production in Streptomyces

Received: 6 March 2002 / Revised: 3 October 2002 / Accepted: 11 October 2002 ' Published online: 9 November 2002
9 Springer-Verlag 2002

Abstract T h e o c c u r r e n c e o f p o l y - 3 - h y d r o x y b u t y r i c acid nutrient-limiting c o n d i t i o n s and m a i n l y serves as a c a r b o n


(PHB) in 12 different strains o f the genus Streptomyces a n d / o r energy storage c o m p o u n d ( S t e i n b u c h e l 1996).
was investigated. Gas c h r o m a t o g r a p h i c e s t i m a t i o n indi- Streptomyces are e x t r e m e l y versatile bacteria c a p a b l e
cated that all the strains p r o d u c e d P H B and the range o f o f synthesizing a variety o f p r i m a r y and s e c o n d a r y
m a x i m u m P H B a c c u m u l a t i o n w a s b e t w e e n 1.5 and 11.8% metabolites, e s p e c i a l l y antibiotics. W h i l e there are pre-
dry cell weight. P H B was i s o l a t e d f r o m Streptomyces l i m i n a r y data r e g a r d i n g the p r o d u c t i o n o f P H B by Strep-
coelicolor A3(2) M145 and c h a r a c t e r i z e d using F o u r i e r tomyces ( K a n n a n and R e h a c e k 1970; M a n n a et al. 1999),
t r a n s f o r m - i n f r a r e d (FT-IR) spectroscopy. The correlation this process has yet to be studied in detail. Two e n z y m e s
b e t w e e n P H B u t i l i z a t i o n a n d a n t i b i o t i c p r o d u c t i o n in o f the P H B b i o s y n t h e t i c pathway, a c e t o a c e t y l - C o A reduc-
S. coelicolor A 3 ( 2 ) M145, was studied; results i n d i c a t e d a tase and [3-ketothiolase, h a v e b e e n identified in Strepto-
p o s s i b l e role o f P H B as a c a r b o n reserve m a t e r i a l used for myces coelicolor (Packter and F l a t m a n 1983). In Strepto-
antibiotic production. myces venezuelae, P H B serves as a m i n o r source o f ace-
t o a c e t y l - C o A for c h l o r a m p h e n i c o l b i o s y n t h e s i s (Ranade
Keywords P o l y h y d r o x y a l k a n o a t e s 9 and Vining 1993). In Streptomyces hygroscopicus, P H B
P o l y - 3 - h y d r o x y b u t y r i c acid 9 Streptomyces 9 d e g r a d a t i o n is thought to p r o v i d e b u t y r y l - C o A during
Gas chromatography - FT-IR spectroscopy 9 p r o d u c t i o n o f the m a c r o l i d e a s c o m y c i n F K 5 2 0 (Wu et al.
3 - H y d r o x y b u t y r i c acid m e t h y l ester 9 A c t i n o r h o d i n 9 2000).
7-Actinorhodin 9Undecylprodigiosin In the present work, we q u a n t i f i e d the p r o d u c t i o n o f
P H B by 12 different strains o f Streptomyces and charac-
terized the p o l y m e r p r o d u c e d by Streptomyces coelicolor
Introduction A3(2) M145. T h e correlation b e t w e e n P H B d e g r a d a t i o n
and antibiotic p r o d u c t i o n was also studied.
P o l y h y d r o x y a l k a n o a t e s ( P H A ) are p o l y e s t e r s o f various
3-, 4-, and 5 - h y d r o x y a l k a n o i c acids that a c c u m u l a t e in
various prokaryotes. O f these, p o l y - 3 - h y d r o x y b u t y r i c acid Materials and methods
(PHB) is one o f the best-studied. P H A exhibit t h e r m o -
plastic properties, are b i o d e g r a d a b l e , and can be p r o d u c e d Growth of bacterial strains
f r o m r e n e w a b l e carbon sources. Hence, there has been The various Streptomyces strains were routinely propagated in
c o n s i d e r a b l e interest in the c o m m e r c i a l e x p l o i t a t i o n o f YEME medium (Kieser et al. 2000). Before standard measurement
these b i o d e g r a d a b l e polyesters. P H B a c c u m u l a t e s under of PHB production, the strains were grown in production medium
(Kannan and Rehacek 1970). S. coelicolor A3(2) M145 was grown
in supplemented minimal media (SMM; Kieser et al, 2000) for the
correlation study. Spore suspensions were used for inoculation in
S. Verma all cases. Dry cell weight was determined after freeze-drying the
Department of Biochemical Engineering and Biotechnology, centrifuged cell mass. In case of residual CaCO3 following the use
Indian Institute of Technology, of production medium,, the wet cell mass was initially treated with
Hauz Khas, New Delhi-110016, India 1 N HCI, washed, and then freeze-dried.

Y. Bhatia 9 S.P. Valappil 9I. Roy (:~)


Department of Biotechnology, School of Biosciences, Isolation, quantitation, and analysis of PHB
University of Westminster,
London W1 W 6UW, United Kingdom The cells were treated according to the method of Law and
e-mail: royi@wmin.ac.uk, Slepecky (1961) and subjected to a soxhlet extraction for 24 h us-
Tel.: + 44 20-79115000 ext.3567, Fax: +44-20-79115087 ing chloroform. The chloroform, containing PHB, was then sub-
67
jected to vacuum rotary evaporation after which PHB remained in
the flask. For FT-IR analysis, the PHB was precipitated from the Results and discussion
chloroform using cold ethanol. PHB was quantitated using a slight
modification of the gas chromatographic method of Braunegg et In order to investigate the a c c u m u l a t i o n o f P H B in the
al. (1978). Instead of whole cells, pure, extracted PHB was used.
For FT-IR analysis, freeze-dried PHB, cells, or precipitated poly- genus Streptornyces, 12 different strains o f Streptomyces
mer was used to prepare KBr discs (sample:KBr, 1:100). An FT-IR were a n a l y z e d for P H B content using gas c h r o m a t o g r a p h y
spectrum 1720X spectrometer (Perkin Elmer, USA) was used un- (Table 1). All 12 strains o f Streptomyces p r o d u c e d PHB,
der the following conditions: spectral range, 4,000-400 cm-I; win- with the m a x i m u m a m o u n t v ar y i n g b e t w e e n 1.5 and
dow material, CsI; 16 scans; resolution 4 cm-l; the detector was a
temperature-stabilized, coated FR-DTGS detector. 11.8% dry cell weight. M a x i m a l P H B p r o d u c t i o n was by
S. coelicolor A3(2) M145 (11.8% dry cell weight). A wide
variation in the P H B co n t en t and the time required for
Measurement of antibiotics produced m a x i m a l P H B production was observed. H o w e v e r , the
by S. coelicolor A3(2) M145
p r o d u c t i o n o f P H B by all o f the strains indicated a spe-
S. coelicolor A3(2) M145 produces, among others, three different cific m e t a b o l i c role o f P H B .
antibiotics, actinorhodin, T-actinorhodin and undecylprodigiosin H a v i n g established that all the strains o f Streptomyces
("red" antibiotic), which were measured separately. Amounts of studied p r o d u c e d P H B , S. coelicolor A3(2) M1 4 5, for
actinorhodin and y-actinorhodin were measured as described in
w h i c h the c o m p l e t e g e n o m e s e q u e n c e is n o w k n o w n , was
Byrstrykh et al. (1996). The amount of undecylprodigiosin was de-
termined as described in Tsao et al. (1986). used for further studies. P H B was isolated f r o m S. coeli-
color A3(2) M145 and ch ar act er i zed using F T- I R spec-
troscopy. H o n g et al. (1999) f o u n d that absorption bands
Chemicals at 1,728 c m -t, c o r r e s p o n d i n g to the ester c a r b o n y l group,
All chemicals were obtained from Merck (Darmstadt, Germany), and at 1,282 c m q , c o r r e s p o n d i n g to the - C H group, are
BDH (Poole, UK) and Sigma (St. Louis, Mo., USA). Soybean meal characteristic o f P H B and can be used to distinguish it
was purchased from Hi-media (Bombay, India). f r o m other P H A s . Th ese characteristic peaks w e r e ob-
s e r v e d for the sample c o n t a i n i n g dried cell pellet (Fig. la),
the precipitated p o l y m e r (Fig. lb), and standard P H B ob-
tained f r o m S i g m a (Fig. lc); T h e FT- I R spectra o f the iso-
lated p o l y m e r and the standard P H B were almost super-

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

Streptomyces strain PHB content


Time of measurement (h)

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

80 b 40 60 80 100 120 140 160 ~80 200


(h)
7o Time

%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
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