Article

Regulation of PHB metabolism in Alcaligenes eutrophus

Canadian Journal of Microbiology (Impact Factor: 1.18). 02/2011; 41(13):44-49. DOI: 10.1139/m95-166

ABSTRACT Metabolites associated with the poly(3-hydroxybutyrate) (PHB) biosynthetic pathway in Alcaligenes eutrophus were measured to gain an insight into the regulation of PHB synthesis in vivo. Alcaligenes eutrophus was grown in carbon-limited chemostat culture to provide bacteria producing negligible PHB, and in nitrogen-limited chemostat culture to yield PHB-synthesizing bacteria. 3-Hydroxybutyryl-CoA (3HBCoA) was detected only in polymer-accumulating bacteria. The level of coenzyme A (CoASH) was approximately three times higher in the absence of PHB synthesis, in accord with the putative role of this metabolite in the regulation of 3-ketothiolase. The level of acetoacetyl-CoA was, however, similar in PHB-accumulating and nonaccumulating bacteria, suggesting that NADPH-acetoacetyl-CoA reductase may regulate PHB synthesis in bacteria grown under carbon limitation. Immediately after nitrogen exhaustion in batch culture of A. eutrophus, there was an initial large decrease in the weight-average molecular weight, which corresponded to the rapid disappearance of CoASH and the maximum level of 3HBCoA. The decrease in the rate of PHB synthesis in batch culture was consistent with regulation involving NADPH-acetoacetyl-CoA reductase. The disappearance of 3HBCoA coincided with the cessation of PHB synthesis and the maximum level of acetyl-CoA.Key words: metabolites, PHB biosynthesis, regulation, Alcaligenes eutrophus, molecular weight.

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    • "The carbon sources are mainly used for the cell growth because the acetyl-CoA produced from the metabolisms of the free fatty acids via β-oxidation pathway flows into the TCA cycle. Besides that, the increase flux of acetyl-CoA into the TCA cycle will enhance the pool of free coenzyme A (CoASH) which will inhibit the 3-ketothiolase that is the first enzyme involved in PHA biosynthesis (Mansfield et al., 1995). Thus the PHA accumulated decreased when high concentration of urea used. "
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    ABSTRACT: Aims: Improper disposal of domestic wastes, such as waste cooking oil (WCO), contributes to the deterioration of the environment and may lead to health problems. In this study, we evaluated the potential of plant-based WCO as a carbon source for the commercial biosynthesis of the bio-plastics, poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). The consumption of WCO for this purpose would mitigate their pollution of the environment at the same time. Methodology and Results: WCO collected from several cafeterias in USM was tested as the carbon source for polyhydroxyalkanoates (PHA) production. A selection of suitable nitrogen source was first conducted in order to obtain an acceptable number of dry cell weight (DCW) and PHA content. Urea was found to be a suitable nitrogen source for the two bacterial strains used in our study, Cupriavidus necator H16 and its transformed mutant, C. necator PHB¯4 harboring the PHA synthase gene of Aeromonas caviae (PHB¯4/pBBREE32d13). With WCO as the sole carbon source, C. necator H16 yielded a relatively good dry cell weight (DCW=25.4 g/L), with 71 wt% poly(3-hydroxybutyrate) P(3HB) content. In comparison, the DCW obtained with fresh cooking oil (FCO) was 24.8 g/L. The production of poly(3 hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from WCO by the transformant C. necator PHB ¯ 4 was comparable, yielding a DCW of 22.3 g/L and P(3HB-co-3HHx) content of 85 wt%. Lipase activities for both bacterial strains reached a maximum after 72 h of cultivation when time profile was conducted. Conclusion, significance and impact of study: The use of WCO as a carbon source in the biosynthesis of the bio-plastic, PHA, turns a polluting domestic waste into a value-added biodegradable product. This renewable source material can thus be exploited for the low cost production of PHA.
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    • "More acetyl-CoA will be channeled to the TCA cycle for cellular metabolism. Besides, the reaction of citrate synthase in TCA cycle also leads to the accumulation of high concentration of free CoASH which is known to inhibit the condensation activity of β-ketothiolase (PhaA) [33]. Therefore, it is necessary to determine the optimal C/N ratio in order to attain high PHA yield. "
    Bioprocess Sciences and Technology, Edited by Min-Tze Liong, 01/2011: pages 55-84; Nova Science Publishers, Inc., USA., ISBN: 978-1-61122-950-9
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    • "In this study, when the concentration of urea was increased to 50 mM, accumulation of PHA copolymer up to 30 wt% could still be observed. Mansfield et al. (1995) found that the level of CoASH increased to maximum during exponential growth phase. Although the CoASH has reached the maximum level, small amounts of P(3HB) were detected, indicating that 3-ketothiolase could not be totally inhibited by CoASH. "
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    ABSTRACT: The combination of plant oils and 3-hydroxyvalerate (3HV) precursors were evaluated for the biosynthesis of polyhydroxyalkanoate (PHA) copolymers containing 3HV monomers by Cupriavidus necator H16. Among various mixtures of plant oils and 3HV-precursors, the mixture of palm kernel oil and sodium propionate was suitable for the biosynthesis of high concentration of PHA (6.8gL(-1)) containing 7mol% of 3HV. The 3HV monomer composition can be regulated in the range of 0-23mol% by changing culture parameters such as the initial pH, and the nitrogen source and its concentration. PHA copolymers with high weight-average molecular weights (Mw) ranging from 1,400,000 to 3,100,000Da were successfully produced from mixtures of plant oils and 3HV-precursors. The mixture of plant oils and sodium propionate resulted in PHA copolymers with higher M(w) compared to the mixture of plant oils and sodium valerate. DSC analysis on the PHA containing 3HV monomers showed the presence of two distinct melting temperature (Tm), which indicated that the PHA synthesized might be a blend of P(3HB) and P(3HB-co-3HV). Sodium propionate appears to be the better precursor of 3HV than sodium valerate.
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