Polyhydroxyalkanoic acids and rhamnolipids are synthesized sequentially in hexadecane fermentation by Pseudomonas aeruginosa ATCC 10145

Rhamnolipids and poly(beta-hydroxyalkanoic acids) (PHAs) are important fermentation products of Pseudomonas aeruginosa. Both contain beta-hydroxyalkanoic acids as main constituents. To investigate the possible relationship between their syntheses, we studied the n-hexadecane fermentation by P. aeruginosa (ATCC 10145). PHA synthesis was found to occur only during active cell growth, while substantial rhamnolipid production began at the onset of the stationary phase. The specific synthesis rate of beta-hydroxyalkanoic acids was estimated as 12.6 mg HA/(g dry cells.h) from the PHA formation during the exponential-growth phase. A similar rate was obtained from the beta-hydroxyalkanoic acid incorporation in the rhamnolipids produced during the early stationary phase. A regulatory switch of the flow of beta-hydroxyalkanoic acids from PHA polymerization to rhamnolipid synthesis is clearly indicated to occur when the culture reaches the stationary phase. Five rhamnolipid structures were identified using HPLC-MS. Three are monorhamnolipids, two dirhamnolipids. All have a chain of two beta-hydroxyalkanoic acids. The two major components contain only beta-hydroxydecanoic acids; the three minors also have a beta-hydroxydecanoic acid linked to the sugar but a beta-hydroxydodecanoic acid or beta-hydroxydodecenoic acid as the second acid. The PHA accumulation reached about 7.5% of the cell dry weight. The monomer composition was relatively constant at different stages of production: in weight fractions, beta-hydroxyoctanoic acid, 0.25 (+/-0.05); beta-hydroxydecanoic acid, 0.41 (+/-0.06); beta-hydroxydodecanoic acid, 0.11 (+/-0.05), beta-hydroxytetradecanoic acid, 0.11 (+/-0.06), and beta-hydroxyhexadecanoic acid, 0.12 (+/-0.06). beta-Hydroxydecanoic acid was clearly the primary monomer.     

Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa

The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924. Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively. Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control. The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly. Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source. Both PHAs and rhamnolipids were synthesized after the growth phase. PHA content in the cell reached a maximum when the carbon source was exhausted. After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued. This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids. The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P. aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids. High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures. High PHA content was achieved in the resting cells that were finally harvested after the second batch. Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves.     

Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads

The fluorescent pseudomonads are classified as a group, one characteristic of which is that they do not accumulate poly-3-hydroxybutyrate (PHB) during nutrient starvation in the presence of excess carbon source. In this paper we show that prototype strains from this subclass, such as Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens, do accumulate poly-3-hydroxyalkanoates (PHA) when grown on fatty acids. These PHAs are composed of medium-chain-length (C6 to C12) 3-hydroxy fatty acids. The ability to form these polyesters does not depend on the presence of plasmids. A specificity profile of the enzymes involved in the biosynthesis of PHA was determined by growing Pseudomonas oleovorans on fatty acids ranging from C4 to C18. In all cases, PHAs were formed which contained C6 to C12 3-hydroxy fatty acids, with a strong preference for 3-hydroxyoctanoate when Ceven fatty acids were supplied and 3-hydroxynonanoate when Codd fatty acids were the substrate. These results indicate that the formation of PHAs depends on a specific enzyme system which is distinct from that responsible for the synthesis of PHB. While the fluorescent pseudomonads are characterized by their inability to make PHB, they appear to share the capacity to produce PHAs. This characteristic may be helpful in classifying pseudomonads. It may also be useful in the optimization of PHA production for biopolymer applications.     

Exploitation of olive oil mill wastewater for combined biohydrogen and biopolymers production

The present study aimed to the investigation of the feasibility of the combined biohydrogen and biopolymers production from OMW (Olive oil Mill Wastewater), using a two stage system. H₂ and volatile fatty acids (VFAs) were produced via anaerobic fermentation and subsequently the acidified wastewater was used as substrate for aerobic biodegradable polymer production. Two different bioreactors, one of CSTR type and a SBR were used for the anaerobic and the aerobic process respectively. The anaerobic reactor was operated at different hydraulic retention times (HRTs) with OMW, diluted 1:4 (v/v) with tap water, as feed. The main VFAs produced were acetate, butyrate and propionate, in different ratios depending on the HRT. Valerate, isovalerate and isobutyrate were also detected in small quantities. Selective effluents of the acidogenic/hydrogen producing reactor were subsequently used as feed for the aerobic reactor. The aerobic reactor was inoculated with an enriched PHAs producing bacteria culture, and was operated in sequential cycles of nitrogen offer (growth phase) and nitrogen limitation (PHAs accumulation phase). The operational program of the SBR was determined according to the results from batch test, and its performance was evaluated for a period of 100 days. During the accumulation phase butyrate was consumed preferably, indicating that the dominant PHA produced is polyhydroxybutyrate. The higher yield of PHAs observed was 8.94% (w/w) of dry biomass weight.     

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.     

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.     

Hyperproduction of polyesters consisting of medium-chain-length hydroxyalkanoate monomers by strain Pseudomonas stutzeri 1317

Pseudomonas stutzeri strain 1317 was found to grow on various fatty acids, alcohols, diols, as well as glucose and gluconate for the synthesis of polyhydroxyalkanoates (PHA) with various monomer units. The PHA monomer structures were dependent on the type of fatty acids and alcohols, as well as the diols in the culture media. Only even number monomers, such as 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD), were accumulated when even numbered fatty acids, alcohols, glucose and gluconate, as well as diol were used as carbon sources. Odd numbered fatty acids and odd numbered alcohols led to the formation of odd numbered monomers, such as 3-hydroxyvalerate (HV), 3-hydroxyheptanoate (HHp), 3-hydroxynonanoate (HN) and 3-hydroxyundecanoate (HU). The strain tolerated up to 1.5% of ethanol and made 8.3% of PHA when growth was conducted in 1.2% of ethanol. PHA formed up to 77% of cell dry weight when the strain was grown in tridecanoate. PHA synthesis was highly dependent on the nitrogen source. A depletion in nitrogen supply immediately resulted in PHA accumulation in cells grown in the glucose mineral medium.     

Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp

A newly acquired polyhydroxyalkanoate (PHA) producing Bacillus spp. was identified to be a strain of Bacillus cereus using a range of microbiological and molecular techniques. This strain, named B. cereus SPV, was found to be capable of using a wide range of carbon sources including glucose, fructose, sucrose, various fatty acids and gluconate for the production of PHAs, an advantage for the commercial production of the polymers. The media used for the polymer production was novel in the context of the genus Bacillus. The PHA, once produced, was found to remain at a constant maximal concentration, without any degradation, a great advantage for the commercial production of the PHAs. This particular strain of Bacillus spp. was able to synthesize various PHAs with 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB)-like monomer units from structurally unrelated carbon sources such as fructose, sucrose and gluconate. This is the first report of the incorporation of a 4HB related monomer containing PHA by the genus Bacillus and from structurally unrelated carbon sources. The PHAs isolated had molecular weights ranging between (0.4 and 0.8) x 10(6) and low polydispersity index values (M(W)/M(N)) ranging from 2.6 to 3.4.     

Synthesis of <Emphasis Type="Italic">scl</Emphasis>-poly (3-hydroxyalkanoates) by <Emphasis Type="Italic">Bacillus cereus</Emphasis> found in freshwater,from monosaccharides and disaccharides

Background

Polyhydroxyalkanoates are a good substitute for synthetic plastic because they are highly biocompatible, ecofriendly, and biodegradable. Bacteria in freshwater bodies such as rivers, tube wells, and canals are exposed to alternating high and low concentrations of substrates that induce PHA production.

Methods

Fresh water samples were collected for isolation of bacterial strains. Screening of PHA in bacterial cells was performed with Sudan and Nile Red staining. Extracted PHA was characterized by FTIR.

Results

In this study, nine bacterial isolates were selected for PHA production on the basis of phenotypic screening. Their ability to accumulate PHAs was determined using different monosaccharides and disaccharides. Two bacterial isolates Bacillus cereus T1 (KY746353) and Bacillus cereus R3 (KY746354) produced PHAs. Optimal growth of the bacterial strain (T1) was observed in the presence of glucose, followed by maximum production of PHAs (63% PHAs) during the logarithmic phase of growth. B. cereus R3 (KY746354) accumulated 60% PHAs by dry cell weight.

Conclusion

PHA accumulation was relatively less with fructose, but both strains showed increased production (up to 50%) with sucrose. The polymer produced was characterized by Fourier-transform infrared spectroscopy (FTIR), which showed that the compound contains short-chain PHAs.

     

Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Direct utilization of palm oil for the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids was demonstrated using Pseudomonas aeruginosa IFO3924. By secreted lipase, palm oil was hydrolyzed into glycerol and fatty acids. Fatty acids became favorable carbon sources for cell growth and PHA production via β-oxidation and glycerol for rhamnolipid production via de novo fatty acid synthesis. Both PHA and rhamnolipid syntheses started after the nitrogen source was exhausted and cell growth ceased. PHA synthesis continued until all fatty acids were exhausted, and at that time, PHA content in the cells reached a maximum, but stopped despite the remaining glycerol (<2g/l). In contrast, rhamnolipid synthesis continued until glycerol was exhausted.     

Cometabolic biosynthesis of copolyesters consisting of 3-hydroxyvalerate and medium-chain-length 3-hydroxyalkanoates by Pseudomonas sp. DSY-82

A newly isolated strain, designated as Pseudomonas sp. DSY-82, synthesized medium-chain-length polyhydroxyalkanoate (MCL-PHA) copolyesters when grown on alkanoates from hexanoate to undecanoate as the sole carbon source. When used alone, butyrate and valerate supported the growth of the isolate but not PHA production. However, unusual polyesters containing 3-hydroxyvalerate, as well as various MCL 3-hydroxyalkanoate monomeric units, were synthesized when valerate was cofed with either nonanoate or 10-undecenoate, suggesting the formation of monomer units from both substrates. Concentrations of 3-hydroxyvalerate, 3-hydroxyoctanoate, and 3-hydroxydecanoate in the PHAs produced were significantly elevated by the addition of valerate, indicating that the incorporation of these monomer units to PHA occurred primarily through cometabolism. The total amount of these monomer units in the PHAs reached up to 30%. The PHAs produced in this study were most likely random copolyesters as determined by differential scanning calorimetric analysis. This is the first case of microbial synthesis of copolyesters consisting of 3-hydroxyvalerate and MCL 3-hydroxyalkanoate monomer units through cometabolism.     

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.     

Advanced bacterial polyhydroxyalkanoates: Towards a versatile and sustainable platform for unnatural tailor-made polyesters

Polyhydroxyalkanoates (PHAs) are biopolyesters that generally consist of 3-, 4-, 5-, and 6-hydroxycarboxylic acids, which are accumulated as carbon and energy storage materials in many bacteria in limited growth conditions with excess carbon sources. Due to the diverse substrate specificities of PHA synthases, the key enzymes for PHA biosynthesis, PHAs with different material properties have been synthesized by incorporating different monomer components with differing compositions. Also, engineering PHA synthases using in vitro-directed evolution and site-directed mutagenesis facilitates the synthesis of PHA copolymers with novel material properties by broadening the spectrum of monomers available for PHA biosynthesis. Based on the understanding of metabolism of PHA biosynthesis, recombinant bacteria have been engineered to produce different types of PHAs by expressing heterologous PHA biosynthesis genes, and by creating and enhancing the metabolic pathways to efficiently generate precursors for PHA monomers. Recently, the PHA biosynthesis system has been expanded to produce unnatural biopolyesters containing 2-hydroxyacid monomers such as glycolate, lactate, and 2-hydroxybutyrate by employing natural and engineered PHA synthases. Using this system, polylactic acid (PLA), one of the major commercially-available bioplastics, can be synthesized from renewable resources by direct fermentation of recombinant bacteria. In this review, we discuss recent advances in the development of the PHA biosynthesis system as a platform for tailor-made polyesters with novel material properties.     

Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses

A three-stage process was developed to produce polyhydroxyalkanoates (PHAs) from sugar cane molasses. The process includes (1) molasses acidogenic fermentation, (2) selection of PHA-accumulating cultures, (3) PHA batch accumulation using the enriched sludge and fermented molasses. In the fermentation step, the effect of pH (5–7) on the organic acids profile and productivity was evaluated. At higher pH, acetic and propionic acids were the main products, while lower pH favoured the production of butyric and valeric acids. PHA accumulation using fermented molasses was evaluated with two cultures selected either with acetate or fermented molasses. The effect of organic acids distribution on polymer composition and yield was evaluated with the acetate selected culture. Storage yields varied from 0.37 to 0.50 Cmmol HA/Cmmol VFA. A direct relationship between the type of organic acids used and the polymers composition was observed. Low ammonia concentration (0.1 Nmmol/l) in the fermented molasses stimulated PHA storage (0.62 Cmmol HA/Cmmol VFA). In addition, strategies of reactor operation to select a PHA-accumulating culture on fermented molasses were developed. The combination of low organic loading with high ammonia concentration selected a culture with a stable storage capacity and with a storage yield (0.59 Cmmol HA/Cmmol VFA) similar to that of the acetate-selected culture.     

Coexpression of genetically engineered 3-ketoacyl-ACP synthase III (fabH) and polyhydroxyalkanoate synthase (phaC) genes leads to short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production from glucose in Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C3 to C5, medium-chain-length (MCL) PHAs consist of monomer units of C6 to C14, and SCL-MCL PHAs consist of monomers ranging in size from C4 to C14. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C4 to C10 and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.     

The role of dissolved oxygen content as a modulator of microbial polyhydroxyalkanoate synthesis

Polyhydroxyalkanoates (PHAs) are a diverse class of bio-polymers synthesized by bacteria, usually during imbalanced growth conditions. Optimizing PHA productivity is highly dependent on the bioreactor oxygen transfer rate (OTR), which is an important consideration for process performance and economics, particularly with increasing scale. Relatively few in-depth studies are available regarding the effect of OTR and dissolved oxygen content (DOC) on PHA formation, synthesis rates, composition, and characteristics. This review examines past research studies on the effect of low DOC environments on production of short-chain length (scl-) PHAs, synthesized by both pure and mixed cultures, in order to identify opportunities and gaps concerning the effect of DOC on production of medium-chain length (mcl-) PHAs, an area that has not been studied in detail. The literature indicates that production of scl-PHA (a reductive process) acts as an electron sink allowing cells to maintain balanced redox state at low DOC. Conversely, production of mcl-PHA via fatty acid de novo synthesis (also a reductive process) does not occur to any significant extent in low DOC environments, while mcl-PHA synthesis from fatty acids (an oxidative process) can be promoted in low DOC environments. The monomer composition, molecular mass, as well as physical and thermal properties of the polymer can change in response to OTR, but further research in this area is required for both scl- and mcl-PHAs. Process design and management of bioreactor OTR in PHA production might therefore be directed by the final application of the polymer rather than cost considerations.     

Production of polyhydroxyalkanoates from agricultural waste and surplus materials

To be competitive with common plastics, the production costs of polyhydroxyalkanoates (PHAs) have to be minimized. Biotechnological polymer production occurs in aerobic processes; therefore, only about 50% of the main carbon sources and even a lower percentage of the precursors used for production of co-polyesters end up in the products wanted. A second cost factor in normally phosphate-limited production processes for PHAs is the costs for complex nitrogen sources. Both cheap carbon sources and cheap nitrogen sources are available from agricultural waste and surplus materials and make a substantial contribution for minimizing PHA production costs. In this study, fermentations for PHA production were carried out in laboratory-scale bioreactors on hydrolyzed whey permeate and glycerol liquid phase from the biodiesel production using a highly osmophilic organism. Without any precursor, the organism produced a poly[3(hydroxybutyrate-co-hydroxyvalerate)] copolyester on both carbon sources. During the accumulation phases, a constant 3-hydroxyvalerate content of 8-10% was obtained at a total PHA concentration of 5.5 g/L (on hydrolyzed whey permeate) and 16.2 g/L (glycerol liquid phase). In an additional fermentation, an expensive nitrogen source was substituted by meat and bone meal beside the glycerol liquid phase as a carbon source, resulting in a final PHA concentration of 5.9 g/L.     

Effective enhancement of short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production by coexpression of genetically engineered 3-ketoacyl-acyl-carrier-protein synthase III (fabH) and polyhydroxyalkanoate synthesis genes

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that have a wide variety of physical properties dependent on the lengths of the pendant groups of the monomer units in the polymer. PHAs composed of mostly short-chain-length (SCL) monomers are often stiff and brittle, whereas PHAs composed of mostly medium-chain-length (MCL) monomers are elastomeric in nature. SCL-MCL PHA copolymers can have properties between the two states, dependent on the ratio of SCL and MCL monomers in the copolymer. It is desirable to elucidate new and low cost ways to produce PHA composed of mostly SCL monomer units with a small mol % of MCL monomers from renewable resources, since this type of SCL-MCL PHA copolymer has superior qualities compared to SCL homopolymer. To address this issue, we have created strains of recombinant E. coli capable of producing beta-ketothiolase (PhbA) and acetoacetyl-CoA synthase (PhbB) from Ralstonia eutropha, genetically engineered 3-ketoacyl-ACP synthase III (FabH) from Escherichia coli, and genetically engineered PHA synthases (PhaC) from Pseudomonas sp. 61-3 to enhance the production of SCL-MCL PHA copolymers from glucose. The cumulative effect of having two monomer-supplying pathways and genetically engineered PHA synthases resulted in higher accumulated amounts of SCL-MCL PHA copolymer from glucose. Polymers were isolated from two recombinant E. coli strains, the first harboring the phbAB, fabH(F87T), and phaC1(SCQM) genes and the second harboring the phbAB, fabH(F87W), and phaC1(SCQM) genes. The thermal and physical properties of the isolated polymers were characterized. It was found that even a very low mol % of MCL monomer in a SCL-MCL PHA copolymer had dramatic effects on the thermal properties of the copolymers.     

The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.