- Email: [email protected]
Biosynthesis of novel copolyesters containing 3-hydroxypivalic acid by Rhodococcus ruber NCIMB 40126 and related bacteria
FEMS Microbiology Letters 159 (1998) 85^92
Biosynthesis of novel copolyesters containing 3-hydroxypivalic acid by Rhodococcus ruber NCIMB 40126 and related bacteria Bernd Fuëchtenbusch, Dirk Fabritius, Marc Waëltermann, Alexander Steinbuëchel * Institut fuër Mikrobiologie der Westfaëlischen Wilhems-Universitaët Muënster, CorrensstraMe 3, D-48149 Muënster, Germany Received 5 December 1997 ; accepted 8 December 1997
Abstract Rhodococcus ruber and related Gram-positive bacteria synthesized and accumulated novel copolyesters containing 3hydroxypivalic acid as constituent if the cells were cultivated in a mineral salts medium containing 3-hydroxypivalic acid and glucose as carbon sources. The copolyesters contributed 0.4^10% of the cellular dry mass, and they contained up to 78 mol% of 3-hydroxypivalic acid in addition to 3-hydroxybutyric acid and 3-hydroxyvaleric acid; a homopolyester of 3-hydroxypivalic acid was also synthesized under certain conditions. The presence of 3-hydroxypivalic acid in the accumulated copolyesters was confirmed by nuclear magnetic resonance spectrometry as well by coupled gas chromatography/mass spectrometry. This is the first time that the incorporation of 3-hydroxypivalic acid and therefore of a hydroxyalkanoic acid with two methyl group substituents at the K-carbon atom in a naturally occurring copolyester is reported. It indicates that 3-hydroxypivalic acidcoenzyme A is accepted by polyhydroxyalkanoic acid synthase as a substrate. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Rhodococcus ruber; 3-Hydroxybutyric acid; 3-Hydroxypivalic acid ; 2,2-Dimethyl-3-hydroxypropionic acid ; Polyhydroxyalkanoic acid
1. Introduction Since the detection of poly(3-hydroxy)butyric acid in 1926 [1], more than 100 other 3-hydroxyalkanoic acids have been detected as constituents of polyhydroxyalkanoic acids (PHA) accumulated by bacteria [2]. These polyesters are stored as insoluble inclusions by various bacteria mostly under growth lim* Corresponding author. Tel.: +49 (251) 8339821; Fax: +49 (251) 8338388; E-mail: [email protected] Abbreviations : PHA, polyhydroxyalkanoic acids ; 3HB, 3-hydroxybutyric acid ; 3HV, 3-hydroxyvaleric acid ; 3HP, 3-hydroxypivalic acid
ited conditions, i.e. if a suitable carbon source is available in excess and if a nutrient essential for growth is lacking. Some of the currently known constituents of bacterial PHA are derived from intermediates of bacterial metabolism if the coenzyme A thioesters of the corresponding hydroxyalkanoic acids are synthesized. However, most constituents were only detected in polyesters, when precursor substrates were fed [3]. The capability of Rhodococcus ruber to accumulate a copolyester consisting of 3hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) in combination with the storage of triacylglycerols has already been reported [4,5]. The genera Rhodococcus and Nocardia are known for their
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 5 4 9 - 1
FEMSLE 7977 26-1-98
86
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
capability to catalyze transformation and degradation of various hydrophobic substances [6]. PHA containing alkyl side chains as constituents at carbon atoms di¡erent from the L-carbon atoms in the polyester backbone promise interesting properties. Recently, we demonstrated that 2-methyl-3-hydroxybutyric acid is incorporated by Ralstonia eutropha (reclassi¢ed from Alcaligenes eutrophus) and Burkholderia cepacia from tiglic acid [7]; this constituent was also found in PHA isolated from environmental samples [8]. In this study we investigated the ability of some strains of the genera Rhodococcus and Nocardia to grow on 3-hydroxypivalic acid and to accumulate PHA containing this component as a new constituent.
2. Materials and methods 2.1. Bacterial strains, media and growth conditions The bacterial strains used in this study are listed in Table 1. The cells were grown under aerobic conditions at 30³C in nutrient broth (NB) or mineral salts medium (MSM) according to Schlegel et al. [9] with di¡erent carbon sources (Table 2). The concentration of ammonium chloride was reduced to 0.05% (w/v) to promote the accumulation of PHA. In some experiments the cells were ¢rst grown in MSM with 0.1% (w/v) ammonium chloride and subsequently transferred to MSM without any nitrogen source. To obtain solid media 1.5% (w/v) agar was added. 3-Hydroxypivalic acid (3-hydroxy-2,2-dimethylpropionic acid) was a generous gift of Dr. H.-J. Traenckner (Bayer AG, Leverkusen). In addition, 3-hydroxypivalic acid purchased from Merck (Darmstadt, Germany) was also used. 2.2. Determination of the 3-hydroxypivalic acid concentration in the growth medium To determine the 3-hydroxypivalic acid concentration in the medium cells were sedimented by centrifugation and the supernatant was lyophilized and subsequently analyzed by gas chromatography as described below.
2.3. Isolation of PHA from lyophilized cells PHA was isolated from lyophilized cells by extraction with chloroform in a Soxhlet apparatus. The polymer was precipitated in 10 volumes of ethanol, and separated from the solution by ¢ltration. After ¢ltration the polymer was dried by exposure to a stream of air. The precipitated polymer was washed with diethylether and precipitated in methanol to remove fatty acids and triacylglycerols. Precipitation and washing was repeated to obtain highly puri¢ed PHA samples. 2.4. Gas chromatographic analysis of PHA For the analysis of PHA 5^7 mg of lyophilized cells or of isolated polyesters were subjected to methanolysis in the presence of 15% sulfuric acid (v/v) suspended in methanol, and the resulting methylesters of hydroxyalkanoic acids were analyzed by gas chromatography according to Brandl et al. [10] and Timm et al. [11] employing a Model 8420 Perkin and Elmer gas chromatograph equipped with a £ame ionization detector and a Permaphase PEG 25 Mx ë berlingen, Gercapillary column (Perkin-Elmer, U many). 2.5. GC/MS analysis The methylesters of the hydrolyzed polyesters were analyzed with coupled gas chromatography/mass spectrometry on a Varian GC 3400 gas chromatograph with a SE 52 capillary column (length 25 m) coupled to a Finnigan MAT 8320 mass spectrometer with data system SS 300. 2.6. NMR analysis The NMR spectra were recorded on a Bruker WM 300 spectrometer. The 300 MHz 1 H-NMR spectra were recorded from a CDCl3 solution of the polyester (25 mg ml31 ) at 20³C, 4.1943 s acquisition time and 3906.25 Hz spectral width. The 13 C-NMR spectra were recorded from a CDCl3 solution of the samples using 1 H-decoupling, 0.3932 s acquisition time and 20833.33 Hz spectral width. 1 H-NMR and 13 C-NMR chemical shifts were referred to
FEMSLE 7977 26-1-98
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
87
CHCl3 and CDCl3 (c = 7.24 ppm and c = 77.0 ppm, respectively).
and 30 h were revealed. If glucose was added as a second substrate, growth was faster and higher cell densities were obtained.
3. Results
3.2. PHA accumulation in strains of the genera Nocardia and Rhodococcus
3.1. Utilization of pivalic acid as a carbon source for growth At the beginning of this study we demonstrated that `Nocardia corallina N³724' (uncertain assignment), R. eutropha H16 and several strains of the genus Rhodococcus (Table 1) were able to grow on MSM agar plates with 3-hydroxypivalic acid as sole carbon source. All strains of Rhodococcus and `N. corallina N³724' exhibited reasonable growth. `N. corallina N³724' and the strains of Rhodococcus formed opaque colonies; staining with Sudan black indicated the accumulation of hydrophobic compounds in the cells of these colonies. In contrast, R. eutropha formed only very small transparent colonies which were not stained with Sudan black, indicating that this bacterium was not a suitable candidate for PHA production from this substrate. When the strains of Rhodococcus sp. and `N. corallina N³724' were cultivated in liquid MSM with 3hydroxypivalic acid as sole carbon source (Fig. 1), doubling times for the optical density between 20
`N. corallina N³724' and the Rhodococcus strains were incubated in nitrogen limited MSM containing 3-hydroxypivalic acid alone or together with other carbon sources. All investigated strains except R. opacus MR 22 accumulated small amounts of PHA consisting of 3HB and 3HV if cultivated with 3-hydroxypivalic acid as sole carbon source (Table 1). Gas chromatographic analysis of the hydroxyalkanoic acid methylesters obtained from whole cells of R. opacus MR 22 revealed in addition to the methylesters of 3HB (retention time: 13.8 min) and 3HV (16.8 min) a compound with a retention time of 15.0 min, which was identical to the methylesters prepared from commercially available 3-hydroxypivalic acid. In R. ruber and R. opacus strain PD630 incorporation of 3HP into PHA was only detected when the cells were cultivated in the presence of 3-hydroxypivalic acid plus glucose (Table 1). Cells of R. eutropha did not accumulate any detectable PHA from 3-hydroxypivalic acid. Carbon sources others than glucose were also
Table 1 PHA accumulation by di¡erent bacterial strains cultivated on 3-hydroxypivalic acid and glucose Bacterial strain (Reference)
Carbon source
Rhodococcus ruber (NCIB 40126, [15]) Rhodococcus opacus PD630 (DSMZ 44193, [16]) Rhodococcus erythropolis (DSMZ 43060) Rhodococcus opacus MR 22 (DMSZ 3346) `Nocardia corallina N³724' [17] Ralstonia eutropha H16 (DSMZ 428)
Glucose+3HP 3HP Glucose+3HP 3HP Glucose+3HP 3HP Glucose+3HP 3HP Glucose+3HP 3HP Gluconate+3HP 3HP
PHA composition
PHA content (in % of CDM)
3HB (mol%)
3HV (mol%)
3HP (mol%)
70.3 67.6 80.0 81.3 79.1 80.8 80.0 77.3 82.9 n.d. 100.0 n.d.
11.1 26.4 16.0 18.7 20.9 19.2 20.0 18.8 17.1 100 n.d. n.d.
18.6 n.d. 4.0 n.d. n.d. n.d. n.d. 3.9 n.d. n.d. n.d. n.d.
6.1 5.3 5.0 4.8 4.8 4.7 5.0 5.3 4.7 9.6 30.5 n.d.
The bacteria were cultivated in MSM containing 0.05% (w/v) NH4 Cl for 60 h at 30³C. 100 ml of medium in 300-ml Erlenmeyer £asks was inoculated with cells grown on NB containing 0.3% 3-hydroxypivalic acid. The concentration of 3-hydroxypivalic acid was 0.3% (w/v) at the beginning; after 24 h and 48 h additional 0.3% (w/v), 0.4% (w/v) 3-hydroxypivalic acid was added, respectively. Glucose or gluconate were added at concentrations of 0.5% (w/v). PHA content and composition were determined by gas chromatography. NCIMB, National Collection of Industrial and Marine Bacteria; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen; n.d., not detected.
FEMSLE 7977 26-1-98
88
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
Fig. 1. Growth of bacteria on glucose and 3-hydroxypivalic acid. The bacterial strains were cultivated in MSM containing 0.5% NH4 Cl (w/v). Growth experiments with open symbols represent experiments in which 0.3% (w/v) 3-hydroxypivalic acid was added as sole carbon source from the beginning with further additions of each 0.2% 3-hydroxypivalic acid after 24 and 48 h. Growth curves with ¢lled symbols represent experiments which 0.3% (w/ v) 3-hydroxypivalic acid plus 0.5% glucose were added as carbon sources from the beginning with further addition of each 0.2% 3hydroxypivalic acid after 24 and 48 h. b,a R. ruber ; F,E R. erythropolis; R,O `N. corallina N³724' ; S,P R. opacus PD630 ; 8,W R. opacus MR 22.
tested as cosubstrates to promote the incorporation of 3-hydroxypivalic acid into PHA by R. ruber (Ta-
ble 2). Mannitol, gluconate, lactose and citrate drastically increased the incorporation of 3-hydroxypivalic acid, with citrate even a homopolyester of 3HP was detected, but the cell densities and the polyester content of the cells were rather low. With acetate as cosubstrate, a polyester content of 24.1% cell dry mass (CDM) was obtained, but 3HP contributed only 1.7 mol% of the constituents. Yeast extract at concentrations up to 1% (w/v) increased the cell densities up to 3.2 g l31 ; however, the PHA content was 0.49% of CDM. The accumulated polyester consisted of 54.5 mol% 3HB, 15.9 mol% 3HV and 25.1 mol% 3HP. 3.3. Accumulation of PHA in the presence of acrylic acid Recently, it was found that R. ruber, cultivated in MSM under nitrogen limited condition, and with valeric acid as carbon source in the presence of 1 mg acrylic acid ml31 , was unable to accumulate triacylglycerols, instead, larger quantities of PHA were accumulated [5]. Acrylic acid inhibits the enzymes acyl-CoA synthase and 3-ketoacyl-CoA thiolase of the L-oxidation pathway [12]. In the presence of acrylic acid (0.2 mg ml31 ) and yeast extract and
Table 2 Composition of PHA in R. ruber cultivated in the presence of 3-hydroxypivalic acid plus di¡erent cosubstrates Carbon source
3HP+glucose 3HP+citrate 3HP+glutarate 3HP+glycerol 3HP+malate 3HP+mannitol 3HP+lactose 3HP+gluconate 3HP+acetate 3HP+yeast extract 3HP+yeast extracta
CDM (g l31 )
Content of polyester (% of CDM)
0.8 0.7 0.3 0.4 0.1 0.4 0.4 1.4 1.2 3.2 3.0
6.1 3.3 9.1 1.9 11.3 8.6 1.6 5.0 24.1 4.9 0.45
PHA composition 3HB (mol%)
3HV (mol%)
3HP (mol%)
70.3 n.d. 73.0 40.0 67.3 15.5 44.7 10.0 21.1 54.5 74.9
11.1 n.d. 13.4 39.4 27.3 9.9 12.9 12.2 77.2 15.9 n.d.
18.6 100.0 13.6 20.6 5.4 74.6 42.4 77.8 1.7 29.6 25.1
a In this experiment 0.02% acrylic acid (w/v) was added. The cells were cultivated in NB for 24 h at 30³C and transferred to MSM containing 0.05% (w/v) NH4 Cl for 50 h at 30³C. 100 ml of medium in 300-ml Erlenmeyer £asks was inoculated. The concentration of 3-hydroxypivalic acid was 0.4% (w/v) at the beginning, after 24 h additional 1% 3-hydroxypivalic acid (w/v) was added. Cosubstrates were added at concentrations of 1% (w/v). PHA content and composition were determined by gas chromatography. Cultivation in the presence of acrylic acid was done as follows : R. ruber was cultivated in NB medium containing 0.3% (w/v) 3-hydroxypivalic acid for 24 h at 30³C, harvested, and resuspended in MSM containing 3-hydroxypivalic acid and glucose as carbon sources for 24 h, when 0.2% (w/v) acrylic acid was added. After a cultivation period of 48 h the cells were harvested.
FEMSLE 7977 26-1-98
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
89
48 h in 5 l of MSM containing 1.0 (w/v) yeast extract and 1% of 3-hydroxypivalic acid (w/v) which were distributed in 500-ml portions in 2-l Erlenmeyer £asks. After 24 h cultivation 0.2 mg acrylic acid ml31 was added, and after an additional cultivation for 24 h the cells were harvested. Approximately 12.6 g cells were obtained, lyophilized and extracted with chloroform. Ethanol precipitation yielded 150 mg PHA with 3HB as major (74.1 mol%) and 3HP (25.9 mol%) as minor constituent according to the gas chromatographic analysis. Fig. 2. Structural formula of poly(3HB-co-3HV).
with 3-hydroxypivalic acid as carbon source R. ruber accumulated small amounts of a copolyester consisting of 3HB and 3HP (0.45% CDM); 3HV was not detected. 3.4. Analysis of the polyester isolated from cells of R. ruber To con¢rm the composition of the polyester accumulated by R. ruber, the cells were cultivated for
3.5. Coupled gas chromatography/mass spectrometry analysis of the hydrolysis products of poly(3-hydroxybutyrate-co-3-hydroxypivalate) The polyester was treated as described before, and the resulting methylesters of the monomers were subjected to coupled gas chromatography/mass spectrometry. The two methyl esters gave the following mass spectra: Compound 1: m/z (%) = 117 (1) [M H], 103 (20) [M -CH3 ], 100 (4) [M -H2 O], 87 (16) [McLa¡erty, C4 H7 O 2 +13], 74 (48) [McLa¡erty, C3 H6 O 2 ], 73 (1) [M -C2 H5 O2 ], 59 (12) [C2 H3 O2 ],
Fig. 3. 1 H-NMR spectrum of poly(3HB-co-3HP) recorded for a polyester isolated from cells of R. ruber.
FEMSLE 7977 26-1-98
90
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
Fig. 4.
13
C-NMR spectrum of poly(3HB-co-3HP) recorded for a polyester isolated from cells of R. ruber.
45 (50) [C2 OH 5 ], 43 (100) [C3 H7 ]; compound 2: m/z (%) = 131 (1) [M -H], 117 (1) [M -CH3 ], 114 (8) [M -H2 O], 102 (100) [M -2CH3 ], 87 (45) [McLa¡ erty, +13. C4 H7 O 2 ], 73 (32) [M -C2 H3 O2 ], 59 (8) [C2 H3 O2 ], 55 (24) [C4 H7 ], 43 (10) [C3 H 7 ]. The data for compound 1 corresponded to those described for 3HB methylester [13], and the data for compound 2 were identical with those obtained from 3HP methylester prepared from commercially available 3HP standard.
3.6. NMR spectroscopic analyses of the polymer To further verify the presence of this new constituent, the PHA sample was also subjected to NMR spectroscopy analysis. The registered peaks contained the signals of carbon atoms of 3HP together with the chemical shifts reported from 3HB containing copolymers [7]. In the 13 C-NMR spectrum the signals of the carbonyl carbons of the polyester split clearly from 169 ppm (a1) to 181 ppm (b1) (Fig. 3, Table 3). The resonance of the monomers at 19.67 (a4), 40.74 (a2), 67.59 (a3), and 169.13 (a1) ppm were assigned by data comparison to 3HB according to
previous reports [14], whereas the resonances at 21.90 (b4 and b5), 43.90 (b3), 69.31 (b2) and 181.96 (b1) ppm were referred to 3HP (Fig. 2). The proton NMR analysis supported the results from the 13 C-NMR analysis: 1 H-NMR of the puri¢ed polymer (CDCl3 ) c (ppm) = 1.22; 1.25; 1.27 (3s, 9H, a4, b3, b5, -CH3 ); 2,45 (dd, 2 J = 15.50 Hz; 3 J = 5.72 Hz; diastereotrop H; a2-Hx); 2.59 (dd, 2 J = 15.50 Hz; 3 J = 7.16 Hz, diastereotrop H; a2Table 3 Chemical shift data from the 3HP)
13
C-NMR spectra of poly(3HB-co-
Repeating unit
Carbon atom
Chemical shifts [in ppm]
3HB
a1 a2 a3 a4
(2q) (t) (d) (q)
169.13 40.74 67.59 19.67
3HP
b1 b2 b3 b4 b5
(s) (s) (t) (2q) (2q)
181.96 69.31 43.90 21.90 21.90
For further details see Fig. 2 and text.
FEMSLE 7977 26-1-98
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
HY ); 3.58 (S, 2H, b2 -CH2 ); 5,24 (dd, 3 J2x;3 = 6.32 Hz; 3 J2y;3 = 13.00 Hz, a3-H) (Fig. 4). 4. Discussion
[2]
[3]
The aim of this study was to demonstrate the incorporation of 3-hydroxypivalic acid into PHA. Among some Gram-positive bacteria investigated, R. ruber NCIMB 40126, R. opacus MR22 and `N. corallina N³724' were able to synthesize the copolyester poly(3HB-co-3HP) or the terpolyester poly(3HB-co-3HV-co-3HP) when the cells were cultivated in the presence of 3-hydroxypivalic acid as carbon source alone or in combination with various cosubstrates. This is the ¢rst report of 3-hydroxypivalic acid as a constituent of bacterial PHA and it was proved by nuclear magnetic resonance spectrometry and coupled gas chromatography/mass spectrometry with commercially available 3-hydroxypivalic acid as standard. The incorporation of 3HP into PHA led to the conclusion that 3HP is accepted as a substrate by a thiokinase or an CoA transferase in these strains resulting in the formation of 3HP-CoA and also that the thioester is used by the PHA synthase as a substrate. The degradation of 3-hydroxypivalic acid has not been studied yet; in addition, the authors are not aware of biogenic 3-hydroxypivalic acid in living matter. But the capability of the bacteria investigated in this study to grow on 3-hydroxypivalic acid as the sole carbon source and to synthesize from it PHA consisting of 3HB and 3HV suggests that these strains degrade 3-hydroxypivalic acid to acetylCoA, and propionyl-CoA.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgments Provision of 3-hydroxypivalic acid by Dr. Traencker (Bayer AG) and support of this study by the Fonds der Chemischen Industrie are gratefully acknowledged.
[14] [15]
References [1] Lemoigne, M. (1926) Produits de deshydration et de polymer-
[16]
91
isation de l'acide L-oxybutyrique. Bull. Soc. Chim. Biol. 8, 770^782. Steinbuëchel, A. and Valentin, H. E. (1995) Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol. Lett. 128, 219^228. Anderson, A.J. and Dawes, E.A. (1990) Occurrence, metabolism, metabolic role and industrial use of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, 450^472. Haywood, G.W., Anderson, A.J., Wiliams D.R., Dawes, E.A. and Ewing, D.F. (1991) Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. Int. J. Biol. Macromol. 13, 83^88. Alvarez, H.M., Kalscheuer, R. and Steinbuëchel, A. (1997) Accumulation of storage lipids in species of Rhodococcus and Nocardia and e¡ect of inhibitors and polyethylene glycol. Fett/Lipid 99, 239^246. Finnerty, W.R. (1992) The biology and genetics of the genus Rhodococcus ruber. Annu. Rev. Microbiol. 46, 193^ 218. Fuëchtenbusch, B., Fabritius, D. and Steinbuëchel, A. (1996) Incorporation of 2-methyl-3-hydroxybutyric acids by axenic cultures in de¢ned media. FEMS Microbiol. Lett. 138, 153^ 160. Satho, H., Mino, T. and Matsuo, T. (1992) Uptake of organic substrates and accumulation of polyhydroxyalkanoates linked with glycolysis of intracellular carbohydrates under anaerobic conditions in the biological excess phosphate removal process. Water Sci. Technol. 26, 933^942. Schlegel, H.G., Kaltwasser, H. and Gottschalk, G. (1961) Ein Submersverfahren zur Kultur wassersto¡oxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch. Mikrobiol. 38, 209^222. Brandl, H., Gross, R.A., Lenz, R.W. and Fuller, R.C. (1988) Pseudomonas oleovorans as a source of poly(L-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl. Environ. Microbiol. 54, 1977^1982. Timm, A., Byrom, D. and Steinbuëchel, A. (1990) Formation of blends of various poly(3-hydroxyalkanoic acids) by a recombinant strain of Pseudomonas oleovorans. Appl. Microbiol. Biotechnol. 33, 296^301. Thijusse, G.J.E. (1964) Fatty acid accumulation by acrylate inhibition of L-oxidation in an alkane-oxidizing Pseudomonas. Biochim. Biophys. Acta 84, 195^197. Doi, Y., Kunioka, M., Nakamura, Y. and Soga, K. (1986) Nuclear magnetic resonance studies on poly(L-hydroxybutyrate) and a copolyester of L-hydroxybutyrate and L-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules 19, 2860^2864. NIST Spectral Database (1992) National Institute of Standards and Technology (USA) Version 4.0. Haywood G.W., Anderson, A.J., Williams, D.R., Dawes, E.A. and Ewing, D.F. (1991) Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. Int. J. Biol. Macromol. 13, 83^88. Alvarez, H.M., Mayer, F., Fabritius, D. and Steinbuëchel, A.
FEMSLE 7977 26-1-98
92
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
(1996) Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630. Arch. Microbiol. 165, 377^368. [17] Valentin, H.E. and Dennis, D. (1996) Metabolic pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) formation in
Nocardia corallina : Inactivation of mutB by chromosomal integration of a kanamycin resistance gene. Appl. Environ. Microbiol. 62, 372^379.
FEMSLE 7977 26-1-98
Biosynthesis of novel copolyesters containing 3-hydroxypivalic acid by Rhodococcus ruber NCIMB 40126 and related bacteria Bernd Fuëchtenbusch, Dirk Fabritius, Marc Waëltermann, Alexander Steinbuëchel * Institut fuër Mikrobiologie der Westfaëlischen Wilhems-Universitaët Muënster, CorrensstraMe 3, D-48149 Muënster, Germany Received 5 December 1997 ; accepted 8 December 1997
Abstract Rhodococcus ruber and related Gram-positive bacteria synthesized and accumulated novel copolyesters containing 3hydroxypivalic acid as constituent if the cells were cultivated in a mineral salts medium containing 3-hydroxypivalic acid and glucose as carbon sources. The copolyesters contributed 0.4^10% of the cellular dry mass, and they contained up to 78 mol% of 3-hydroxypivalic acid in addition to 3-hydroxybutyric acid and 3-hydroxyvaleric acid; a homopolyester of 3-hydroxypivalic acid was also synthesized under certain conditions. The presence of 3-hydroxypivalic acid in the accumulated copolyesters was confirmed by nuclear magnetic resonance spectrometry as well by coupled gas chromatography/mass spectrometry. This is the first time that the incorporation of 3-hydroxypivalic acid and therefore of a hydroxyalkanoic acid with two methyl group substituents at the K-carbon atom in a naturally occurring copolyester is reported. It indicates that 3-hydroxypivalic acidcoenzyme A is accepted by polyhydroxyalkanoic acid synthase as a substrate. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Rhodococcus ruber; 3-Hydroxybutyric acid; 3-Hydroxypivalic acid ; 2,2-Dimethyl-3-hydroxypropionic acid ; Polyhydroxyalkanoic acid
1. Introduction Since the detection of poly(3-hydroxy)butyric acid in 1926 [1], more than 100 other 3-hydroxyalkanoic acids have been detected as constituents of polyhydroxyalkanoic acids (PHA) accumulated by bacteria [2]. These polyesters are stored as insoluble inclusions by various bacteria mostly under growth lim* Corresponding author. Tel.: +49 (251) 8339821; Fax: +49 (251) 8338388; E-mail: [email protected] Abbreviations : PHA, polyhydroxyalkanoic acids ; 3HB, 3-hydroxybutyric acid ; 3HV, 3-hydroxyvaleric acid ; 3HP, 3-hydroxypivalic acid
ited conditions, i.e. if a suitable carbon source is available in excess and if a nutrient essential for growth is lacking. Some of the currently known constituents of bacterial PHA are derived from intermediates of bacterial metabolism if the coenzyme A thioesters of the corresponding hydroxyalkanoic acids are synthesized. However, most constituents were only detected in polyesters, when precursor substrates were fed [3]. The capability of Rhodococcus ruber to accumulate a copolyester consisting of 3hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) in combination with the storage of triacylglycerols has already been reported [4,5]. The genera Rhodococcus and Nocardia are known for their
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 5 4 9 - 1
FEMSLE 7977 26-1-98
86
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
capability to catalyze transformation and degradation of various hydrophobic substances [6]. PHA containing alkyl side chains as constituents at carbon atoms di¡erent from the L-carbon atoms in the polyester backbone promise interesting properties. Recently, we demonstrated that 2-methyl-3-hydroxybutyric acid is incorporated by Ralstonia eutropha (reclassi¢ed from Alcaligenes eutrophus) and Burkholderia cepacia from tiglic acid [7]; this constituent was also found in PHA isolated from environmental samples [8]. In this study we investigated the ability of some strains of the genera Rhodococcus and Nocardia to grow on 3-hydroxypivalic acid and to accumulate PHA containing this component as a new constituent.
2. Materials and methods 2.1. Bacterial strains, media and growth conditions The bacterial strains used in this study are listed in Table 1. The cells were grown under aerobic conditions at 30³C in nutrient broth (NB) or mineral salts medium (MSM) according to Schlegel et al. [9] with di¡erent carbon sources (Table 2). The concentration of ammonium chloride was reduced to 0.05% (w/v) to promote the accumulation of PHA. In some experiments the cells were ¢rst grown in MSM with 0.1% (w/v) ammonium chloride and subsequently transferred to MSM without any nitrogen source. To obtain solid media 1.5% (w/v) agar was added. 3-Hydroxypivalic acid (3-hydroxy-2,2-dimethylpropionic acid) was a generous gift of Dr. H.-J. Traenckner (Bayer AG, Leverkusen). In addition, 3-hydroxypivalic acid purchased from Merck (Darmstadt, Germany) was also used. 2.2. Determination of the 3-hydroxypivalic acid concentration in the growth medium To determine the 3-hydroxypivalic acid concentration in the medium cells were sedimented by centrifugation and the supernatant was lyophilized and subsequently analyzed by gas chromatography as described below.
2.3. Isolation of PHA from lyophilized cells PHA was isolated from lyophilized cells by extraction with chloroform in a Soxhlet apparatus. The polymer was precipitated in 10 volumes of ethanol, and separated from the solution by ¢ltration. After ¢ltration the polymer was dried by exposure to a stream of air. The precipitated polymer was washed with diethylether and precipitated in methanol to remove fatty acids and triacylglycerols. Precipitation and washing was repeated to obtain highly puri¢ed PHA samples. 2.4. Gas chromatographic analysis of PHA For the analysis of PHA 5^7 mg of lyophilized cells or of isolated polyesters were subjected to methanolysis in the presence of 15% sulfuric acid (v/v) suspended in methanol, and the resulting methylesters of hydroxyalkanoic acids were analyzed by gas chromatography according to Brandl et al. [10] and Timm et al. [11] employing a Model 8420 Perkin and Elmer gas chromatograph equipped with a £ame ionization detector and a Permaphase PEG 25 Mx ë berlingen, Gercapillary column (Perkin-Elmer, U many). 2.5. GC/MS analysis The methylesters of the hydrolyzed polyesters were analyzed with coupled gas chromatography/mass spectrometry on a Varian GC 3400 gas chromatograph with a SE 52 capillary column (length 25 m) coupled to a Finnigan MAT 8320 mass spectrometer with data system SS 300. 2.6. NMR analysis The NMR spectra were recorded on a Bruker WM 300 spectrometer. The 300 MHz 1 H-NMR spectra were recorded from a CDCl3 solution of the polyester (25 mg ml31 ) at 20³C, 4.1943 s acquisition time and 3906.25 Hz spectral width. The 13 C-NMR spectra were recorded from a CDCl3 solution of the samples using 1 H-decoupling, 0.3932 s acquisition time and 20833.33 Hz spectral width. 1 H-NMR and 13 C-NMR chemical shifts were referred to
FEMSLE 7977 26-1-98
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
87
CHCl3 and CDCl3 (c = 7.24 ppm and c = 77.0 ppm, respectively).
and 30 h were revealed. If glucose was added as a second substrate, growth was faster and higher cell densities were obtained.
3. Results
3.2. PHA accumulation in strains of the genera Nocardia and Rhodococcus
3.1. Utilization of pivalic acid as a carbon source for growth At the beginning of this study we demonstrated that `Nocardia corallina N³724' (uncertain assignment), R. eutropha H16 and several strains of the genus Rhodococcus (Table 1) were able to grow on MSM agar plates with 3-hydroxypivalic acid as sole carbon source. All strains of Rhodococcus and `N. corallina N³724' exhibited reasonable growth. `N. corallina N³724' and the strains of Rhodococcus formed opaque colonies; staining with Sudan black indicated the accumulation of hydrophobic compounds in the cells of these colonies. In contrast, R. eutropha formed only very small transparent colonies which were not stained with Sudan black, indicating that this bacterium was not a suitable candidate for PHA production from this substrate. When the strains of Rhodococcus sp. and `N. corallina N³724' were cultivated in liquid MSM with 3hydroxypivalic acid as sole carbon source (Fig. 1), doubling times for the optical density between 20
`N. corallina N³724' and the Rhodococcus strains were incubated in nitrogen limited MSM containing 3-hydroxypivalic acid alone or together with other carbon sources. All investigated strains except R. opacus MR 22 accumulated small amounts of PHA consisting of 3HB and 3HV if cultivated with 3-hydroxypivalic acid as sole carbon source (Table 1). Gas chromatographic analysis of the hydroxyalkanoic acid methylesters obtained from whole cells of R. opacus MR 22 revealed in addition to the methylesters of 3HB (retention time: 13.8 min) and 3HV (16.8 min) a compound with a retention time of 15.0 min, which was identical to the methylesters prepared from commercially available 3-hydroxypivalic acid. In R. ruber and R. opacus strain PD630 incorporation of 3HP into PHA was only detected when the cells were cultivated in the presence of 3-hydroxypivalic acid plus glucose (Table 1). Cells of R. eutropha did not accumulate any detectable PHA from 3-hydroxypivalic acid. Carbon sources others than glucose were also
Table 1 PHA accumulation by di¡erent bacterial strains cultivated on 3-hydroxypivalic acid and glucose Bacterial strain (Reference)
Carbon source
Rhodococcus ruber (NCIB 40126, [15]) Rhodococcus opacus PD630 (DSMZ 44193, [16]) Rhodococcus erythropolis (DSMZ 43060) Rhodococcus opacus MR 22 (DMSZ 3346) `Nocardia corallina N³724' [17] Ralstonia eutropha H16 (DSMZ 428)
Glucose+3HP 3HP Glucose+3HP 3HP Glucose+3HP 3HP Glucose+3HP 3HP Glucose+3HP 3HP Gluconate+3HP 3HP
PHA composition
PHA content (in % of CDM)
3HB (mol%)
3HV (mol%)
3HP (mol%)
70.3 67.6 80.0 81.3 79.1 80.8 80.0 77.3 82.9 n.d. 100.0 n.d.
11.1 26.4 16.0 18.7 20.9 19.2 20.0 18.8 17.1 100 n.d. n.d.
18.6 n.d. 4.0 n.d. n.d. n.d. n.d. 3.9 n.d. n.d. n.d. n.d.
6.1 5.3 5.0 4.8 4.8 4.7 5.0 5.3 4.7 9.6 30.5 n.d.
The bacteria were cultivated in MSM containing 0.05% (w/v) NH4 Cl for 60 h at 30³C. 100 ml of medium in 300-ml Erlenmeyer £asks was inoculated with cells grown on NB containing 0.3% 3-hydroxypivalic acid. The concentration of 3-hydroxypivalic acid was 0.3% (w/v) at the beginning; after 24 h and 48 h additional 0.3% (w/v), 0.4% (w/v) 3-hydroxypivalic acid was added, respectively. Glucose or gluconate were added at concentrations of 0.5% (w/v). PHA content and composition were determined by gas chromatography. NCIMB, National Collection of Industrial and Marine Bacteria; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen; n.d., not detected.
FEMSLE 7977 26-1-98
88
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
Fig. 1. Growth of bacteria on glucose and 3-hydroxypivalic acid. The bacterial strains were cultivated in MSM containing 0.5% NH4 Cl (w/v). Growth experiments with open symbols represent experiments in which 0.3% (w/v) 3-hydroxypivalic acid was added as sole carbon source from the beginning with further additions of each 0.2% 3-hydroxypivalic acid after 24 and 48 h. Growth curves with ¢lled symbols represent experiments which 0.3% (w/ v) 3-hydroxypivalic acid plus 0.5% glucose were added as carbon sources from the beginning with further addition of each 0.2% 3hydroxypivalic acid after 24 and 48 h. b,a R. ruber ; F,E R. erythropolis; R,O `N. corallina N³724' ; S,P R. opacus PD630 ; 8,W R. opacus MR 22.
tested as cosubstrates to promote the incorporation of 3-hydroxypivalic acid into PHA by R. ruber (Ta-
ble 2). Mannitol, gluconate, lactose and citrate drastically increased the incorporation of 3-hydroxypivalic acid, with citrate even a homopolyester of 3HP was detected, but the cell densities and the polyester content of the cells were rather low. With acetate as cosubstrate, a polyester content of 24.1% cell dry mass (CDM) was obtained, but 3HP contributed only 1.7 mol% of the constituents. Yeast extract at concentrations up to 1% (w/v) increased the cell densities up to 3.2 g l31 ; however, the PHA content was 0.49% of CDM. The accumulated polyester consisted of 54.5 mol% 3HB, 15.9 mol% 3HV and 25.1 mol% 3HP. 3.3. Accumulation of PHA in the presence of acrylic acid Recently, it was found that R. ruber, cultivated in MSM under nitrogen limited condition, and with valeric acid as carbon source in the presence of 1 mg acrylic acid ml31 , was unable to accumulate triacylglycerols, instead, larger quantities of PHA were accumulated [5]. Acrylic acid inhibits the enzymes acyl-CoA synthase and 3-ketoacyl-CoA thiolase of the L-oxidation pathway [12]. In the presence of acrylic acid (0.2 mg ml31 ) and yeast extract and
Table 2 Composition of PHA in R. ruber cultivated in the presence of 3-hydroxypivalic acid plus di¡erent cosubstrates Carbon source
3HP+glucose 3HP+citrate 3HP+glutarate 3HP+glycerol 3HP+malate 3HP+mannitol 3HP+lactose 3HP+gluconate 3HP+acetate 3HP+yeast extract 3HP+yeast extracta
CDM (g l31 )
Content of polyester (% of CDM)
0.8 0.7 0.3 0.4 0.1 0.4 0.4 1.4 1.2 3.2 3.0
6.1 3.3 9.1 1.9 11.3 8.6 1.6 5.0 24.1 4.9 0.45
PHA composition 3HB (mol%)
3HV (mol%)
3HP (mol%)
70.3 n.d. 73.0 40.0 67.3 15.5 44.7 10.0 21.1 54.5 74.9
11.1 n.d. 13.4 39.4 27.3 9.9 12.9 12.2 77.2 15.9 n.d.
18.6 100.0 13.6 20.6 5.4 74.6 42.4 77.8 1.7 29.6 25.1
a In this experiment 0.02% acrylic acid (w/v) was added. The cells were cultivated in NB for 24 h at 30³C and transferred to MSM containing 0.05% (w/v) NH4 Cl for 50 h at 30³C. 100 ml of medium in 300-ml Erlenmeyer £asks was inoculated. The concentration of 3-hydroxypivalic acid was 0.4% (w/v) at the beginning, after 24 h additional 1% 3-hydroxypivalic acid (w/v) was added. Cosubstrates were added at concentrations of 1% (w/v). PHA content and composition were determined by gas chromatography. Cultivation in the presence of acrylic acid was done as follows : R. ruber was cultivated in NB medium containing 0.3% (w/v) 3-hydroxypivalic acid for 24 h at 30³C, harvested, and resuspended in MSM containing 3-hydroxypivalic acid and glucose as carbon sources for 24 h, when 0.2% (w/v) acrylic acid was added. After a cultivation period of 48 h the cells were harvested.
FEMSLE 7977 26-1-98
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
89
48 h in 5 l of MSM containing 1.0 (w/v) yeast extract and 1% of 3-hydroxypivalic acid (w/v) which were distributed in 500-ml portions in 2-l Erlenmeyer £asks. After 24 h cultivation 0.2 mg acrylic acid ml31 was added, and after an additional cultivation for 24 h the cells were harvested. Approximately 12.6 g cells were obtained, lyophilized and extracted with chloroform. Ethanol precipitation yielded 150 mg PHA with 3HB as major (74.1 mol%) and 3HP (25.9 mol%) as minor constituent according to the gas chromatographic analysis. Fig. 2. Structural formula of poly(3HB-co-3HV).
with 3-hydroxypivalic acid as carbon source R. ruber accumulated small amounts of a copolyester consisting of 3HB and 3HP (0.45% CDM); 3HV was not detected. 3.4. Analysis of the polyester isolated from cells of R. ruber To con¢rm the composition of the polyester accumulated by R. ruber, the cells were cultivated for
3.5. Coupled gas chromatography/mass spectrometry analysis of the hydrolysis products of poly(3-hydroxybutyrate-co-3-hydroxypivalate) The polyester was treated as described before, and the resulting methylesters of the monomers were subjected to coupled gas chromatography/mass spectrometry. The two methyl esters gave the following mass spectra: Compound 1: m/z (%) = 117 (1) [M H], 103 (20) [M -CH3 ], 100 (4) [M -H2 O], 87 (16) [McLa¡erty, C4 H7 O 2 +13], 74 (48) [McLa¡erty, C3 H6 O 2 ], 73 (1) [M -C2 H5 O2 ], 59 (12) [C2 H3 O2 ],
Fig. 3. 1 H-NMR spectrum of poly(3HB-co-3HP) recorded for a polyester isolated from cells of R. ruber.
FEMSLE 7977 26-1-98
90
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
Fig. 4.
13
C-NMR spectrum of poly(3HB-co-3HP) recorded for a polyester isolated from cells of R. ruber.
45 (50) [C2 OH 5 ], 43 (100) [C3 H7 ]; compound 2: m/z (%) = 131 (1) [M -H], 117 (1) [M -CH3 ], 114 (8) [M -H2 O], 102 (100) [M -2CH3 ], 87 (45) [McLa¡ erty, +13. C4 H7 O 2 ], 73 (32) [M -C2 H3 O2 ], 59 (8) [C2 H3 O2 ], 55 (24) [C4 H7 ], 43 (10) [C3 H 7 ]. The data for compound 1 corresponded to those described for 3HB methylester [13], and the data for compound 2 were identical with those obtained from 3HP methylester prepared from commercially available 3HP standard.
3.6. NMR spectroscopic analyses of the polymer To further verify the presence of this new constituent, the PHA sample was also subjected to NMR spectroscopy analysis. The registered peaks contained the signals of carbon atoms of 3HP together with the chemical shifts reported from 3HB containing copolymers [7]. In the 13 C-NMR spectrum the signals of the carbonyl carbons of the polyester split clearly from 169 ppm (a1) to 181 ppm (b1) (Fig. 3, Table 3). The resonance of the monomers at 19.67 (a4), 40.74 (a2), 67.59 (a3), and 169.13 (a1) ppm were assigned by data comparison to 3HB according to
previous reports [14], whereas the resonances at 21.90 (b4 and b5), 43.90 (b3), 69.31 (b2) and 181.96 (b1) ppm were referred to 3HP (Fig. 2). The proton NMR analysis supported the results from the 13 C-NMR analysis: 1 H-NMR of the puri¢ed polymer (CDCl3 ) c (ppm) = 1.22; 1.25; 1.27 (3s, 9H, a4, b3, b5, -CH3 ); 2,45 (dd, 2 J = 15.50 Hz; 3 J = 5.72 Hz; diastereotrop H; a2-Hx); 2.59 (dd, 2 J = 15.50 Hz; 3 J = 7.16 Hz, diastereotrop H; a2Table 3 Chemical shift data from the 3HP)
13
C-NMR spectra of poly(3HB-co-
Repeating unit
Carbon atom
Chemical shifts [in ppm]
3HB
a1 a2 a3 a4
(2q) (t) (d) (q)
169.13 40.74 67.59 19.67
3HP
b1 b2 b3 b4 b5
(s) (s) (t) (2q) (2q)
181.96 69.31 43.90 21.90 21.90
For further details see Fig. 2 and text.
FEMSLE 7977 26-1-98
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
HY ); 3.58 (S, 2H, b2 -CH2 ); 5,24 (dd, 3 J2x;3 = 6.32 Hz; 3 J2y;3 = 13.00 Hz, a3-H) (Fig. 4). 4. Discussion
[2]
[3]
The aim of this study was to demonstrate the incorporation of 3-hydroxypivalic acid into PHA. Among some Gram-positive bacteria investigated, R. ruber NCIMB 40126, R. opacus MR22 and `N. corallina N³724' were able to synthesize the copolyester poly(3HB-co-3HP) or the terpolyester poly(3HB-co-3HV-co-3HP) when the cells were cultivated in the presence of 3-hydroxypivalic acid as carbon source alone or in combination with various cosubstrates. This is the ¢rst report of 3-hydroxypivalic acid as a constituent of bacterial PHA and it was proved by nuclear magnetic resonance spectrometry and coupled gas chromatography/mass spectrometry with commercially available 3-hydroxypivalic acid as standard. The incorporation of 3HP into PHA led to the conclusion that 3HP is accepted as a substrate by a thiokinase or an CoA transferase in these strains resulting in the formation of 3HP-CoA and also that the thioester is used by the PHA synthase as a substrate. The degradation of 3-hydroxypivalic acid has not been studied yet; in addition, the authors are not aware of biogenic 3-hydroxypivalic acid in living matter. But the capability of the bacteria investigated in this study to grow on 3-hydroxypivalic acid as the sole carbon source and to synthesize from it PHA consisting of 3HB and 3HV suggests that these strains degrade 3-hydroxypivalic acid to acetylCoA, and propionyl-CoA.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgments Provision of 3-hydroxypivalic acid by Dr. Traencker (Bayer AG) and support of this study by the Fonds der Chemischen Industrie are gratefully acknowledged.
[14] [15]
References [1] Lemoigne, M. (1926) Produits de deshydration et de polymer-
[16]
91
isation de l'acide L-oxybutyrique. Bull. Soc. Chim. Biol. 8, 770^782. Steinbuëchel, A. and Valentin, H. E. (1995) Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol. Lett. 128, 219^228. Anderson, A.J. and Dawes, E.A. (1990) Occurrence, metabolism, metabolic role and industrial use of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, 450^472. Haywood, G.W., Anderson, A.J., Wiliams D.R., Dawes, E.A. and Ewing, D.F. (1991) Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. Int. J. Biol. Macromol. 13, 83^88. Alvarez, H.M., Kalscheuer, R. and Steinbuëchel, A. (1997) Accumulation of storage lipids in species of Rhodococcus and Nocardia and e¡ect of inhibitors and polyethylene glycol. Fett/Lipid 99, 239^246. Finnerty, W.R. (1992) The biology and genetics of the genus Rhodococcus ruber. Annu. Rev. Microbiol. 46, 193^ 218. Fuëchtenbusch, B., Fabritius, D. and Steinbuëchel, A. (1996) Incorporation of 2-methyl-3-hydroxybutyric acids by axenic cultures in de¢ned media. FEMS Microbiol. Lett. 138, 153^ 160. Satho, H., Mino, T. and Matsuo, T. (1992) Uptake of organic substrates and accumulation of polyhydroxyalkanoates linked with glycolysis of intracellular carbohydrates under anaerobic conditions in the biological excess phosphate removal process. Water Sci. Technol. 26, 933^942. Schlegel, H.G., Kaltwasser, H. and Gottschalk, G. (1961) Ein Submersverfahren zur Kultur wassersto¡oxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch. Mikrobiol. 38, 209^222. Brandl, H., Gross, R.A., Lenz, R.W. and Fuller, R.C. (1988) Pseudomonas oleovorans as a source of poly(L-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl. Environ. Microbiol. 54, 1977^1982. Timm, A., Byrom, D. and Steinbuëchel, A. (1990) Formation of blends of various poly(3-hydroxyalkanoic acids) by a recombinant strain of Pseudomonas oleovorans. Appl. Microbiol. Biotechnol. 33, 296^301. Thijusse, G.J.E. (1964) Fatty acid accumulation by acrylate inhibition of L-oxidation in an alkane-oxidizing Pseudomonas. Biochim. Biophys. Acta 84, 195^197. Doi, Y., Kunioka, M., Nakamura, Y. and Soga, K. (1986) Nuclear magnetic resonance studies on poly(L-hydroxybutyrate) and a copolyester of L-hydroxybutyrate and L-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules 19, 2860^2864. NIST Spectral Database (1992) National Institute of Standards and Technology (USA) Version 4.0. Haywood G.W., Anderson, A.J., Williams, D.R., Dawes, E.A. and Ewing, D.F. (1991) Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. Int. J. Biol. Macromol. 13, 83^88. Alvarez, H.M., Mayer, F., Fabritius, D. and Steinbuëchel, A.
FEMSLE 7977 26-1-98
92
B. Fuëchtenbusch et al. / FEMS Microbiology Letters 159 (1998) 85^92
(1996) Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630. Arch. Microbiol. 165, 377^368. [17] Valentin, H.E. and Dennis, D. (1996) Metabolic pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) formation in
Nocardia corallina : Inactivation of mutB by chromosomal integration of a kanamycin resistance gene. Appl. Environ. Microbiol. 62, 372^379.
FEMSLE 7977 26-1-98