A thermostable #x003B2;-ketothiolase of polyhydroxyalkanoates (PHAs) in <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: Purification and biochemical properties

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a #x003B2;-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 °C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. #x003B2;-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 M and 25 M for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of #x003B2;-ketothiolase. (Mol Cell Biochem 269: 27–36, 2005)     

Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (M_w: 480,000 g.mol^–1) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (M_w: 391,000 g mol^–1) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a -ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a K_m of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70°C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 M CoA and lost all of its activity when it was treated with alkaline phosphatase. PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.     

Cloning and expression of the PHA synthase genes phaC1 and phaC1AB into Bacillus subtilis

Summary Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB. The two plasmids were inserted into Bacillus subtilis DB104 and generated Bacillus subtilis/pBE2C1 and Bacillus subtilis/pBE2C1AB. The two recombinant strains were subjected to fermentation and showed PHA accumulation, the first reported example of medium-chain-length-PHA production in Bacillus subtilis. GC analysis identified the compound produced by Bacillus subtilis/pBE2C1 was a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer while that produced by Bacillus subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. The results also showed that the recombinant B. subtilis could utilize the malt waste in the medium as a carbon source better than that of glucose and thus could substantially lower the cost of production of PHA.     

Effect of the applied organic load rate on biodegradable polymer production by mixed microbial cultures in a sequencing batch reactor

This article studies the operation of a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) at different applied organic load rates (OLRs). The process is based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. A mixture of acetic, lactic, and propionic acids at different concentrations (in the range 8.5-31.25 gCOD/L) was fed every 2 h in a sequencing batch reactor (SBR). The resulting applied OLR was in the range 8.5-31.25 gCOD/L/day. Even though, as expected, the increase in the OLR caused an increase in biomass concentration (up to about 8.7 g COD/L), it also caused a relevant decrease of maximal polymer production rate. This decrease in polymer production rate was related to the different extent of "feast and famine" conditions, as function of the applied OLR and of the start-up conditions. As a consequence the best performance of the process was obtained at an intermediate OLR (20 gCOD/L/day) where both biomass productivity and PHA storage were high enough. However, at this high OLR the process was unstable and sudden decrease of performance was also observed. The sludge characterized by the highest PHA storage response was investigated by 16S rDNA clone library. The clone library contained sequences mostly from PHA producers (e.g., Alcaligenes and Comamonas genera); however many genera and among them, one of the dominant (Thauera), were never described before in relation to PHA storage response.     

Production of polyhydroxyalkanoates (PHAs) from waste materials and by-products by submerged and solid-state fermentation

Polyhydroxyalkanoates are biodegradable polymers produced by prokaryotic organisms from renewable resources. The production of PHAs by submerged fermentation processes has been intensively studied over the last 30 years. In recent years, alternative strategies have been proposed, such as the use of solid-state fermentation or the production of PHAs in transgenic plants. This paper gives an overview of submerged and solid-state fermentation processes used to produce PHAs from waste materials and by-products. The use of these low-cost raw materials has the potential to reduce PHA production costs, because the raw material costs contribute a significant part of production costs in traditional PHA production processes.     

Comparative analysis of PHAs production by Bacillus megaterium OUAT 016 under submerged and solid-state fermentation

In view of risk coupled with synthetic polymer waste, there is an imperative need to explore biodegradable polymer. On account of that, six PHAs producing bacteria were isolated from mangrove forest and affilated to the genera Bacillus & Pseudomonas from morpho-physiological characterizations. Among which the potent PHAs producer was identified as Bacillus megaterium OUAT 016 by 16S rDNA sequencing and in-silico analysis. This research addressed a comparative account on PHAs production by submerged and solid-state fermentation pertaining to different downstream processing. Here, we established higher PHAs production by solid-state fermentation through sonication and mono-solvent extraction. Using modified MSM media under optimized conditions, 49.5% & 57.7% of PHAs were produced in submerged and 34.1% & 62.0% in solid-state fermentation process. Extracted PHAs was identified as a valuable polymer PHB-co-PHV and its crystallinity & thermostability nature was validated by FTIR, ¹H NMR and XRD. The melting (Tm) and thermal degradation temperature (Td) of PHB-co-PHV was 166 °C and 273 °C as depicted from DTA. Moreover, FE-SEM and SPM surface imaging indicated biodegradable nature, while FACS assay confirmed cytocompatibility of PHB-co-PHV.     

Exploring two-stage fermentation strategy of polyhydroxyalkanoate production using Aeromonas hydrophila

With consideration of sustainable development, this study explored the fermentation strategy of cost-effective production of biodegradable polymer- polyhydroxyalanoates (PHAs) for feasibility of eco-friendly materials recycling during wastewater treatment. As prior studies showed that Aeromonas hydrophila NIU01 was a promising PHA-producing bacterium, this follow-up study tended to seek for optimal nutrient-supplementation strategy to stimulate maximal PHA accumulation of A. hydrophila NIU01 for cellular production. As maximal PHA production took place at growth-limiting conditions, two-stage fermentation was much more appropriate for practical applications compared to batch mode of operation. Moreover, this optimal two-stage operation strategy maximized cellular PHA production under nitrogen-limiting conditions at C/N molar ratio of 60/1. For materials recycling, this operation strategy could be applicable to simultaneous PHB production and wastewater decolorization using A. hydrophila.     

Microbial degradation of polyhydroxyalkanoates in tropical soils

The integrated study addressing biodegradation of microbial linear polyesters of hydroxyalkanoic acids (polyhydroxyalkanoates, PHAs) in tropical conditions by microbial communities of Vietnamese soils was performed in locations close to Hanoi and Nha Trang, which differed in their weather conditions and microbial communities. It shows that PHA degradation in tropical soils is influenced by polymer chemical composition, specimen shape, and microbial characteristics. The homopolymer of 3-hydroxybutyric acid is degraded at higher rates than the copolymer of 3-hydroxybutyric and 3-hydroxyvaleric acids. The average rates of mass loss were 0.04–0.33% per day for films and 0.02–0.18% for compact pellets. PHA degradation was accompanied by a decrease in the polymer molecular mass and, usually, an increase in the degree of crystallinity, suggesting preferential degradation of the amorphous phase. Under the study conditions, representatives of the bacterial genera Burkholderia, Bacillus, Cupriavidus, Mycobacterium, and Nocardiopsis and such micromycetes as Acremonium, Gongronella, Paecilomyces, and Penicillium, Trichoderma have been identified as major PHA degraders.     

Recent strategies for efficient production of polyhydroxyalkanoates by micro‐organisms

Polyhydroxyalkanoates (PHAs) are polyesters accumulated by many bacteria under unbalanced growth conditions, and have been used to meet the various demands in areas of agriculture, medicine, and materials especially belong to a rapidly expanding area of biomedical research. Unfortunately, the high production cost than the traditional synthetic materials has greatly limited the wide application of PHA. Here, we systematically summarized recent progress in production of PHAs and a series of optimization strategies such as supplying renewable carbon substrates, developing better bacterial strains, optimization of fermentation processes, engineering new pathways and etc., were applied to reduce production cost, therefore providing many new ideas and methods for the production of PHAs in economically viable processes. This is believed to be a comprehensive report to show different strategies and methods for low‐cost production of PHAs. Further studies are still needed to make PHAs more and more economically viable to meet a wide range of applicability.