PHA Accumulation Research Articles

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.

Characterization, seasonal occurrence, and diel fluctuation of poly(hydroxyalkanoate) in photosynthetic microbial mats.

In situ poly(hydroxyalkanoate) (PHA) levels and repeating-unit compositions were examined in stratified photosynthetic microbial mats from Great Sippewissett Salt Marsh, Mass., and Ebro Delta, Spain. Unlike what has been observed in pure cultures of phototrophic bacteria, the prevalence of hydroxyvalerate (HV) repeating units relative to hydroxybutyrate (HB) repeating units was striking. In the cyanobacteria-dominated green material of Sippewissett mats, the mole percent ratio of repeating units was generally 1HB:1HV. In the purple sulfur bacteria-dominated pink material the relationship was typically 1HB:2HV. In Sippewissett mats, PHA contributed about 0.5 to 1% of the organic carbon in the green layer and up to 6% in the pink layer. In Ebro Delta mats, PHA of approximately 1HB:2HV-repeating-unit distribution contributed about 2% of the organic carbon of the composite photosynthetic layers (the green and pink layers were not separated). Great Sippewissett Salt Marsh mats were utilized for more extensive investigation of seasonal, diel, and exogenous carbon effects. When the total PHA content was normalized to organic carbon, there was little seasonal variation in PHA levels. However, routine daily variation was evident at all sites and seasons. In every case, PHA levels increased during the night and decreased during the day. This phenomenon was conspicuous in the pink layer, where PHA levels doubled overnight. The daytime declines could be inhibited by artificial shading. Addition of exogenous acetate, lactate, and propionate induced two- to fivefold increases in the total PHA levels when applied in the daylight but had no effect when applied at night. The distinct diel pattern of in situ PHA accumulation at night appears to be related, in some phototrophs, to routine dark energy metabolism and is not influenced by the availability of organic nutrients.

PhaG-mediated synthesis of Poly(3-hydroxyalkanoates) consisting of medium-chain-length constituents from nonrelated carbon sources in recombinant Pseudomonas fragi.

Recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxy-alkanoate) consisting of medium-chain-length constituents (C(6) to C(14)) (PHA(MCL)), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylase (PhaG), has been identified in Pseudomonas putida (B. H. A. Rehm, N. Krüger, and A. Steinbüchel, J. Biol. Chem. 273:24044-24051, 1998). To establish this PHA-biosynthetic pathway in a non-PHA-accumulating bacterium, we functionally coexpressed phaC1 (encoding PHA synthase 1) from Pseudomonas aeruginosa and phaG (encoding the transacylase) from P. putida in Pseudomonas fragi. The recombinant strains of P. fragi were cultivated on gluconate as the sole carbon source, and PHA accumulation to about 14% of the total cellular dry weight was achieved. The respective polyester was isolated, and GPC analysis revealed a weight average molar mass of about 130,000 g mol(-1) and a polydispersity of 2.2. The PHA was composed mainly (60 mol%) of 3-hydroxydecanoate. These data strongly suggested that functional expression of phaC1 and phaG established a new pathway for PHA(MCL) biosynthesis from nonrelated carbon sources in P. fragi. When fatty acids were used as the carbon source, no PHA accumulation was observed in PHA synthase-expressing P. fragi, whereas application of the beta-oxidation inhibitor acrylic acid mediated PHA(MCL) accumulation. The substrate for the PHA synthase PhaC1 is therefore presumably directly provided through the enzymatic activity of the transacylase PhaG by the conversion of (R)-3-hydroxydecanoyl-ACP to (R)-3-hydroxydecanoyl-CoA when the organism is cultivated on gluconate. Here we demonstrate for the first time the establishment of PHA(MCL) synthesis from nonrelated carbon sources in a non-PHA-accumulating bacterium, employing fatty acid de novo biosynthesis and the enzymes PhaG (a transacylase) and PhaC1 (a PHA synthase).

Use of metabolic inhibitors and gas chromatography/mass spectrometry to study poly- β-hydroxyalkanoates metabolism involving cryptic nutrients in enhanced biological phosphorus removal systems

The role of the tricarboxylic (TCA) cycle and the glyoxylate pathway in anaerobic accumulation of poly- β-hydroxyalkanoates by enhanced biological phosphorus removal (EBPR) activated sludge was investigated. Accumulation of both poly- β-hydroxybutyrate (PHB) and poly- β-hydroxyvalerate (PHV) was observed, after feeding acetate to fresh activated sludge collected from a pilot-scale EBPR facility. Based on the supplied amount of carbon nutrient the level of PHA synthesis exceeded the level of expected synthesis. Feeding of pyruvate combined with acetate resulted in accumulation of PHB and PHV, in approximately a 1:1 ratio. Citrate addition did not result in PHA accumulation but both PHB and PHV were formed when citrate combined with acetate was fed to the sludge. Succinate or succinate combined with acetate stimulated 2-fold higher accumulation of PHV than PHB. In most cases of synthesis, total PHA accumulation exceeded total carbon input. This excess accumulation suggests the presence of nutrients that are normally unusable or cryptic in the absence of additional complementary nutrients such as short chain fatty acids. This hypothesis is consistent with the fragmentation patterns produced during gas chromatography-mass spectrometric (GC/MS) analyses of PHA extracted from sludge fed with 13C-labeled nutrients. Treating the EBPR sludge by adding different nutrients combined with malonate, α-ketoglutarate and monofluoroacetate, three common inhibitors of steps in the TCA cycle significantly impaired PHA accumulation and implied that these steps can be important for PHA synthesis. A biochemical model that incorporates elements of the TCA cycle and the glyoxylate pathway can account for the observed patterns of PHA accumulation.

Inactivation of isocitrate lyase leads to increased production of medium-chain-length poly(3-hydroxyalkanoates) in Pseudomonas putida.

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two- to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant.

Production of poly(hydroxyalkanoate) by a composite anaerobic acidification–fermentation system

The production of Poly(hydroxyalkanoate) (PHA) from organic wastewater by a composite anaerobic acidification–fermentation system was developed and the degree of acidification and products distribution were examined. The production of PHA from acidified wastewater was examined in both batch and fed-batch fermentation. Butyric acid was the most suitable acid for Alcaligenes eutrophus growth and PHA accumulation. Over 99% acidification and favourable organic acid distribution in the effluent of an acidogenic UASB was obtained at 40°C and pH 5.7. The amount of PHA accumulated in A. eutrophus on the acidified and concentrated wastewater in fed-batch fermentation was 10 g/l, accounting for 63.3% of cell mass.

Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic.

Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.

Production of medium-chain-length poly(3-hydroxyalkanoates) from gluconate by recombinant Escherichia coli.

It was shown recently that recombinant Escherichia coli, defective in the beta-oxidation cycle and harboring a medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) polymerase-encoding gene of Pseudomonas, is able to produce MCL PHA from fatty acids but not from sugars or gluconate (S. Langenbach, B. H. A. Rehm, and A. Steinbüchel, FEMS Microbiol. Lett. 150:303-309, 1997; Q. Ren, Ph.D. thesis, ETH Zürich, Zürich, Switzerland, 1997). In this study, we report the formation of MCL PHA from gluconate by recombinant E. coli. By introduction of genes coding for an MCL PHA polymerase and the cytosolic thioesterase I ('thioesterase I) into E. coli JMU193, we were able to engineer a pathway for the synthesis of MCL PHA from gluconate. We used two expression systems, i.e., the bad promoter and alk promoter, for the 'thioesterase I- and PHA polymerase-encoding genes, respectively, which enabled us to modulate their expression independently over a range of inducer concentrations, which resulted in a maximum MCL PHA accumulation of 2.3% of cell dry weight from gluconate. We found that the amount of PHA and the 'thioesterase I activity are directly correlated. Moreover, the polymer accumulated in the recombinant E. coli consisted mainly of 3-hydroxyoctanoate monomers. On the basis of our data, we propose an MCL PHA biosynthesis pathway scheme for recombinant E. coli JMU193, harboring PHA polymerase and 'thioesterase I, when grown on gluconate, which involves both de novo fatty acid synthesis and beta-oxidation.

Synthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa: comparison of PhaC1 and PhaC2

In order to obtain functional expression of PHA synthase gene phaC2 from Pseudomonas aeruginosa in Escherichia coli, the coding region of phaC2 was subcloned, including the ribosomal binding site, into pBluescript SK − collinear to the lac promoter. This plasmid pBHR71-C2 enabled functional expression of phaC2 in E. coli LS1298 ( fadB) under lac promoter control, leading to PHA accumulation, when grown in LB medium containing 0.5% (w/v) of various fatty acids (C 8-C 14). The strongest accumulation of PHA was observed, when dodecanoate was provided as carbon source, and PHA contributed to 15% of cell dry weight, which was composed of 35 mol% 3-hydroxydodecanoate, 60 mol% 3-hydroxydecanoate and 5 mol% 3-hydroxyoctanoate. Plasmid pBHR78, which contained both genes phaC1 and phaC2 from P. aeruginosa under lac promoter control in pBluescript SK − led in E. coli LS1298 to PHA accumulation, which contributed to 13% of cell dry weight, when cells were grown on decanoate. Only slight differences in PHA composition compared with either PhaC1 or PhaC2 were obtained. The weight average molecular masses of PHA purified from decanoate-grown cells of E. coli LS1298 expressing PhaC1 or PhaC2 alone or both PHA synthases, were 106×10 3, 70×10 3 or 67×10 3, respectively. This study clearly demonstrated that both PHA synthases from P. aeruginosa exhibit very similar properties resulting in similar extent of PHA accumulation, similar composition and molecular mass, when expressed in E. coli and that fatty acid β-oxidation provides substrates for both PHA synthases.

Enhanced yield and a high production of medium-chain-length poly(3-hydroxyalkanoates) in a two-step fed-batch cultivation of Pseudomonas putida by combined use of glucose and octanoate

Production of medium-chain-length (MCL) poly(3-hydroxyalkanoates) (PHAs) from a strain of Pseudomonas putida was studied. Although octanoate was found to be efficient for MCL PHA production, its yield for PHAs was low since it was also used for cell growth. A two-step fed-batch cultivation was thus designed with glucose and octanoate as the main carbon source for cell growth and PHA accumulation, respectively. After the cells were grown on glucose in the first step, they were subjected to nitrogen- and oxygen-limiting conditions in the second step in which octanoate was supplied as the sole carbon source. PHA (18.6 g l −1) was obtained with a yield of about 40% (g PHA g −1 octanoate). By using the same method but supplying octanoate in the first cell-growing step, production of MCL PHAs was significantly enhanced; it yielded 35.9 g PHA l −1 after 39 h of the fed-batch operation. The PHA content of the cell was also improved to 65.5% (g PHA g −1 cell mass) which indicated that octanoate addition stimulated quite efficiently the biosynthesis of MCL PHAs.

Poly(β-hydroxyalkanoate) accumulation in bacterioplankton from Lake Cisó (Spain)

The specific poly(β-hydroxyalkanoate) (PHA) content of the bacterioplankton from Lake Cisó (Spain) was measured at different depths. Phototrophic bacteria reach large populations in this lake and the PHA concentration changed seasonally according to their biomass. During summer stratification of the lake, phototrophic bacteria formed a metalimnetic peak. Bacterial counts were maximal at a depth of 1.5–2.0 m, whereas the PHA specific content reached a maximal value at 3.0–3.5 m. Since the limiting factor in this ecosystem is light availability, we assume that cells beneath the dense metalimnetic peak have unbalanced growth conditions and thus accumulate large amounts of PHA.Key words: bacterioplankton, PHA accumulation, Lake Cisó, light limitation.

Accumulation of PHA and its copolyesters by Methylobacterium sp. KCTC 0048

Methylobacterium sp. KCTC 0048 isolated from soil, could synthesize a variety of copolyesters when secondary carbon substrates were added to nitrogen-limited cultures containing methanol as a major carbon and energy source. The copolyester of 3-hydroxy-butyrate and 3-hydroxyvalerate, P(3HB-co-3HV) accumulated when valeric acid, pentanol or heptanoic acid was added to the nitrogen-limited medium containing methanol. The copolyester of 3-hydroxybutyrate and 4-hydroxybutyrate, P(3HB-co-4HB) was synthesized from 4-hydroxybutyrate, 1,4-butanediol, or γ-butyrolactone, and the copolyester of 3-hydroxybutyrate and 3-hydroxypropionate (P(3HB-co-3HP)), from 3-hydroxypropionate as the secondary carbon substrates, respectively.

Poly(hydroxyalkanoate) in cyanobacteria: an overview

In this paper an overview is given on the occurrence of poly(hydroxyalkanoate) (PHA) in cyanobacteria and its possible role as a putative reserve compound. Comparisons are made with the function of other storage compounds that occur in cyanobacteria. For the cyanobacteria Oscillatoria limosa and Gloeothece sp. PCC 6909, some experimental data on the accumulation and mobilization of PHA are presented. O. limosa presumably contains poly(hydroxyvalerate) (PHV), whereas in Gloeothece poly(hydroxybutyrate) (PHB) was detected. Both species accumulated PHA to 6–9% of the dry weight. In Gloeothece PHB accumulation was stimulated by the addition of acetate but in O. limosa this was not the case. PHA was not involved in dark metabolism in either of the strains.

Poly(hydroxyalkanoate) in cyanobacteria: an overview

In this paper an overview is given on the occurrence of poly(hydroxyalkanoate) (PHA) in cyanobacteria and its possible role as a putative reserve compound. Comparisons are made with the function of other storage compounds that occur in cyanobacteria. For the cyanobacteria Oscillatoria limosa and Gloeothece sp. PCC 6909, some experimental data on the accumulation and mobilization of PHA are presented. O. limosa presumably contains poly(hydroxyvalerate) (PHV), whereas in Gloeothece poly(hydroxybutyrate) (PHB) was detected. Both species accumulated PHA to 6–9% of the dry weight. In Gloeothece PHB accumulation was stimulated by the addition of acetate but in O. limosa this was not the case. PHA was not involved in dark metabolism in either of the strains.

Cyclo-oxygenase and oxygen free radical-stimulated fibroblast proliferation

There was no evidence of accumulation of PHA in any particular organ although trichloroacetic acid-precipitable and total counts (not shown) were high in kidneys, particularly when calculated on a tissue weight basis. Only the liver of tomato-lectin-treated rats contained as much radioactivity as that from PHA-intubated animals. It has been shown previously (Pusztai et al., 1986) that the toxicity of dietary PHA is dependent not just on its local disruptive effects on the small intestine but also on ensuing systemic toxic responses to the lectin. This gives an indirect indication that dietary PHA or other lectins might breach the gut absorptive barrier in appreciable quantities. The results presented (Table 1) are, however, the first direct experimental evidence to support the existence of appreciable systemic absorption of a dietary lectin. It is particularly significant that under the same experimental conditions only a small proportion of the non-toxic tomato lectin is absorbed systemically. This was apparently not due to differential survival of the lectins in the gut, but was more probably due to differences in their binding specificity to and extent of endocytosis by small intestinal cells. As recently shown, even lectins less toxic than PHA gain access to the body by passing through the gut wall and thus are good immunogens by the oral route (de Aizapurua & Russell-Jones, 1988). In a recent review on oral drug delivery (Pusztai, 1989) the potential effectiveness of lectins for such an application was explored. The results in the present paper go a long way to underpin such theoretical considerations and form a basis for practical exploration of this potential.