• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1217-1226
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• 2004

A psychrotrophic bacterial strain, Pseudomonas fluorescens BM07, synthesized unsaturated fatty acids (UFA) from fructose in response to lowering of growth temperature, and incorporated them into both polyhydroxyalkanoic acid (PHA) and membrane lipid. The blocking of PHA synthesis by adding 5 mM 2-bromooctanoic acid to the growth medium, containing 70 mM fructose, was found to be a useful means to profile the composition of membrane lipid by gas chromatography. As the growth temperature changed from 35 to $50^{\circ}C$, the total content of two UFA, 3-hydroxy-cis-5­dodecenoic acid ($C_{12:1}$) and 3-hydroxy-cis-7-tetradecenoic acid ($C_{14:1}$), in PHA increased from 31 to 44 $mol\%$. The growth at lower temperatures also led to an increase in the level of two major UFA, palmitoleic acid (C16:1 cis9) and cis-vaccenic acid (C18:1 cis11), in membrane lipid. A fraction of these membrane-lipid UFA was converted to their corresponding cyclopropane fatty acids (CFA). The CFA conversion was a function of culture time, exhibiting biphasic increase before and after entering the stationary phase. However, pH changes in growth media had no effect on the CFA conversion, which is contrary to the case of E. coli reported. The cells grown at $30^{\circ}C$ responded to a cold shock (lowering the medium temperature down to $10^{\circ}C$) by increasing the level of C16:1 cis9 and C 18: I cis II up to that of $10^{\circ}C$-grown control cells and concomitantly decreasing the relative level of cis-9,10­methylenehexadecanoic acid (the CFA converted from C16:1 cis9) from 14 to 8 $mol\%$, whereas the 10-grown cells exhibited little change in the lipid composition when exposed to a warmer environment of $30^{\circ}C$ for 12 h. Based on this one- way response, we suggest that this psychrotrophic strain responds more efficiently and sensitively to a cold shock than to a hot shock. It is also suggested that BM07 strain is a good producer of two unsaturated 3-hydroxyacids, $C_{12:1}\;and\;C_{141:1}$.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.715-717
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• 2005

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.718-720
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• 2005

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1256-1266
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• 2004

2-Bromooctanoic acid (2-BrOA) is known to block the formation of polyhydroxyalkanoic acid (PHA) in Pseudomonasfluorescens BM07 without any influence on the cell growth when grown on fructose, but it inhibits the cell growth when grown on octanoate (OA) (Lee et al., Appl. Environ. Microbiol. 67: 4963- 4974, 2001). We investigated the role of 2-BrOA in the PHA synthesis of the bacterium grown with mixtures of fructose and fatty acids. OA, 11­phenoxyundecanoic acid (1 1-POU), and 5-phenylvaleric acid (5-PV) were selected as model substrates. When supplemented with 50 mM fructose, all these carboxylic acids suppressed the formation of PHA from fructose, however, the ~-oxidation coenzyme A monomers derived from the carboxylic acids were efficiently polymerized, but the conversion yield [(mol of carboxylate substrate converted into PHA)/(mol of carboxylate substrate in the feed)] was low (e.g., maximally $\~53\%$ for 5 mM 11-POU). Addition of 2-BrOA (up to 5 mM) to the mixed carbon sources raised the conversion yield sensitively and effectively only at low levels of the acid substrates (e.g., 2 mM 1 1-POU or 5 mM OA): For instance, $100\%$ of 2 mM ll-POU were converted into PHA in the presence of 5 mM 2-BrOA, whereas only $\~10\%$ of the 1 1-POU were converted in the absence of 2-BrOA. However, at highly saturated suppressing levels (e.g., 5 mM ll-POU), 2-BrOA inhibitor showed no significant additional effect on the conversion ($60- 70\%$ conversion irrespective of 2-BrOA level). The existence of competitive and compensative relationship between 2­BrOA and all the carboxylic acid substrates used may indicate 'Present address: Section on Brain Physiology and Metabolism, Bldg. 10, Rm. 6N202, National Institute on Agmg, National Institute of Health, Bethesda, MD 20892, U.S.A. that all the acid substrate-derived inhibiting species bind to the same site as the 2-BrOA inhibiting species does. We, therefore, suggest that 2-BrOA can be used for efficiently increasing the yield of conversion of expensive substituted fatty acids into PHA and then substituted 3-hydroxyacids by hydrolyzing it.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1330-1336
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• 2005

Polyhydroxyalkanoic acid (PHA) inclusion bodies were analyzed in situ by $^{13}C$-nuclear magnetic resonance ($^{13}C$-NMR) spectroscopy. The PHA inclusion bodies studied were composed of poly(3-hydroxybutyrate) or poly(3hydroxybutyrate-co-4-hydroxybutyrate), which was accumulated in Hydrogenophaga pseudoflava, and medium-chain-length PHA (MCL-PHA), which was accumulated in Pseudomonas fluorescens BM07 from octanoic acid or 11-phenoxyundecanoic acid (11-POU). The quantification of the $^{13}C$-NMR signals was conducted against a standard compound, sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). The chemical shift values for the in vivo NMR spectral peaks agreed well with those for the corresponding purified PHA polymers. The intracellular degradation of the PHA inclusions by intracellular PHA depolymerase(s) was monitored by in vivo NMR spectroscopy and analyzed in terms of first-order reaction kinetics. The H. pseudoflava cells were washed for the degradation experiment, transferred to a degradation medium without a carbon source, but containing 1.0 g/l ammonium sulfate, and cultivated at $35^{\circ}C$ for 72 h. The in vivo NMR spectra were obtained at $70^{\circ}C$ for the short-chain-length PHA cells whereas the spectra for the aliphatic and aromatic MCL-PHA cells were obtained at $50^{\circ}C\;and\;80^{\circ}C$, respectively. For the H. pseudoflava cells, the in vivo NMR kinetics analysis of the PHA degradation resulted in a first-order degradation rate constant of 0.075/h ($r^{2}$=0.94) for the initial 24 h of degradation, which was close to the 0.050/h determined when using a gas chromatographic analysis of chloroform extracts of sulfuric acid/methanol reaction mixtures of dried whole cells. Accordingly, it is suggested that in vivo $^{13}C$-NMR spectroscopy is an important tool for studying intracellular PHA degradation in terms of kinetics.