Regulation of PHB metabolism in Alcaligenes eutrophus
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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ABSTRACT: It has been shown that Pseudomonas putida GPo1 is able to grow in continuous culture simultaneously limited by ammonium (N source) and octanoate (C source), and concomitantly accumulate poly([R]-3-hydroxyalkanoate) (PHA). Under such growth conditions the material properties of PHA can be fine-tuned if a second PHA precursor substrate is supplied. To determine the range of dual carbon and nitrogen (C, N)-limited growth conditions, tedious chemostat experiments need to be carried out for each carbon source separately. To determine the growth regime, the C/N ratio of the feed (f) to a chemostat was changed in a stepwise manner at a constant dilution rate of 0.3/h. Dual-(C, N)-limited growth was observed between C(f) /N(f) ≤ 6.4 g/g and C(f) /N(f) >9.5 g/g. In the following, we analyzed alternative approaches, using continuous medium gradients at the same dilution rate, that do not require time consuming establishments of steady states. Different dynamic approaches were selected in which the C(f) /N(f) ratio was changed continuously through a convex increase of C(f) , a convex increase of N(f) , or a linear decrease of C(f) (gradients 1, 2, and 3, respectively). In these experiments, the dual-(C, N)-limited growth regime was between 7.2 and 11.0 g/g for gradient 1, 4.3 and 6.9 g/g for gradient 2, and 5.1 and 8.9 g/g for gradient 3. A mathematical equation was developed that compensated a time delay of the gradient that was caused by the wash-in/wash-out effects of the medium feed.Biotechnology Journal 06/2011; 6(10):1240-52. · 3.45 Impact Factor
- BioEssays 03/2011; 33(5):350-7. · 5.42 Impact Factor
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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.