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.     

Production of medium chain length polyhydroxyalkanoates by fed-batch culture of Pseudomonas oleovorans

Pseudomonas oleovorans was cultivated to produce medium chain length polyhydroxyalkanoates (MCL-PHAs) from octanoic acid and ammonium nitrate as carbon and nitrogen source, respectively, by a pH-stat fed-batch culture technique. The octanoate in the culture broth was maintained below 4 g l^–1 by feeding the mixture of octanoic acid and ammonium nitrate when the culture pH rose above 7.1. The final cell concentrations of 63, 55 and 9.5 g l^–1, PHA contents of 62, 75 and 67% of dry cell wt, and productivities of 1, 0.63 and 0.16 g l^–1 h^–1 were obtained when the C/N ratios in the feed were 10, 20 and 100 g octanoic acid g^–1 ammonium nitrate, respectively.     

Slowly utilized carbon sources enhance botulinic specific toxicity by co-culture of Clostridium argentinense with the non-pathogen Pseudomonas mendocina

The growth and botulinic toxin production of Clostridium argentinense G 89 HT in co-culture with Pseudomonas mendocina were studied using two culture systems with carbon sources of rapid and slow utilization. Growth with glucose in homogeneous co-culture presented a dual-phase progression, with the toxin produced in the slow-growing phase. The extended 50 h growth period of the second phase at low specific growth rate was attributed to the combined metabolization of glucose and a complex carbon source of the alginate type produced by P. mendocina under strongly reducing conditions. With dextrin, the co-culture grew at the lower specific growth rate (μ = 0.03 h⁻¹) for a period lasting 80 h. This fully enhanced the production of toxin with a specific toxicity 2.5 times higher than with glucose in a homogeneous system and 10.7 higher than that of C. argentinense G 89 HT single culture. The heterogeneous co-culture obtained with a dialysis membrane physically separating both bacteria (thereby eliminating the metabolic interaction) produced the lowest levels of growth and toxin of all the cases analyzed.     

Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads

Pseudomonas aeruginosa PAO and 15 other strains of this species synthesized a polyester with 3-hydroxydecanoate as the main constituent (55 to 76 mol%) if the cells were cultivated in the presence of gluconate and if the nitrogen source was exhausted; 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydodecanoate were minor constituents of the polymer. The polymer was deposited in granules within the cell and amounted to 70% of the cell dry matter in some strains. Among 55 different strains of 41 Pseudomonas species tested, P. aureofaciens (21.6% of cellular dry matter), P. citronellolis (78.0%), P. chlororaphis (8.5%), P. marginalis (11.4%), P. mendocina (50.7%), P. putida (33.5%), and Pseudomonas sp. strain DSM 1650 (54.6%) accumulated this type of polymer at significant levels (greater than 5%) during cultivation on gluconate. In two strains of P. facilis and P. fluorescens, as well as in one strain of P. syringae, this polymer was detected as a minor constituent (much less than 5%). All other strains accumulated either poly(3-hydroxybutyrate) or a polymer consisting mainly of 3-hydroxyoctanoate with octanoate but no polyester with gluconate as the carbon source. Only a few species (e.g., P. stutzeri) were unable to accumulate poly(hydroxyalkanoic acids) (PHA) at all. These results indicated that the formation of PHA depends on a pathway which is distinct from all other known PHA-biosynthetic pathways. The polyesters accumulated by gluconate- or octanoate-grown cells of recombinant strains of P. aeruginosa and P. putida, which harbored the Alcaligenes eutrophus poly(3-hydroxybutyrate)biosynthetic genes, contained 3-hydroxybutyrate as an additional constituent.     

Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads.

Pseudomonas aeruginosa PAO and 15 other strains of this species synthesized a polyester with 3-hydroxydecanoate as the main constituent (55 to 76 mol%) if the cells were cultivated in the presence of gluconate and if the nitrogen source was exhausted; 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydodecanoate were minor constituents of the polymer. The polymer was deposited in granules within the cell and amounted to 70% of the cell dry matter in some strains. Among 55 different strains of 41 Pseudomonas species tested, P. aureofaciens (21.6% of cellular dry matter), P. citronellolis (78.0%), P. chlororaphis (8.5%), P. marginalis (11.4%), P. mendocina (50.7%), P. putida (33.5%), and Pseudomonas sp. strain DSM 1650 (54.6%) accumulated this type of polymer at significant levels (greater than 5%) during cultivation on gluconate. In two strains of P. facilis and P. fluorescens, as well as in one strain of P. syringae, this polymer was detected as a minor constituent (much less than 5%). All other strains accumulated either poly(3-hydroxybutyrate) or a polymer consisting mainly of 3-hydroxyoctanoate with octanoate but no polyester with gluconate as the carbon source. Only a few species (e.g., P. stutzeri) were unable to accumulate poly(hydroxyalkanoic acids) (PHA) at all. These results indicated that the formation of PHA depends on a pathway which is distinct from all other known PHA-biosynthetic pathways. The polyesters accumulated by gluconate- or octanoate-grown cells of recombinant strains of P. aeruginosa and P. putida, which harbored the Alcaligenes eutrophus poly(3-hydroxybutyrate)biosynthetic genes, contained 3-hydroxybutyrate as an additional constituent.     

Biosynthesis of copolyesters consisting of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids from 1,3-butanediol or from 3-hydroxybutyrate by Pseudomonas sp. A33

Pseudomonas sp. A33 and other isolates of aerobic bacteria accumulated a complex copolyester containing 3-hydroxybutyric acid (3HB) and various medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from 3-hydroxybutyric acid or from 1,3-butanediol under nitrogen-limitated culture conditions. 3HB contributed to 15.1 mol/100 mol of the constituents of the polyester depending on the strain and on the cultivation conditions. The accumulated polymer was a copolyester of 3HB and 3HA_MCL rather than a blend of poly(3HB) and poly(3HA_MCL) on the basis of multiple evidence. 3-Hydroxyhexadecenoic acid and 3-hydroxyhexadecanoic acid were detected as constituents of polyhydroxyalkanoates, which have hitherto not been described, by¹³C nuclear magnetic resonance or by gas chromatography/mass spectrometric analysis. In total, ten different constituents were detected in the polymer synthesized from 1,3-butanediol by Pseudomonas sp. A33:besides seven saturated (3HB, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydrohexadecanoate) three unsaturated (3-hydroxydodecenoate, 3-hydroxytetradecenoate and 3-hydrohexadecanoate) hydroxyalkanoic acid constituents occured. The polyhydroxyalkanoate synthase of Pseudomonas sp. A33 was cloned, and its substrate specificity was evaluated by heterologous expression in various strains of P. putida, P. oleovorans and Alcaligenes eutrophus.     

A lower specificity PhaC2 synthase from Pseudomonas stutzeri catalyses the production of copolyesters consisting of short-chain-length and medium-chain-length 3-hydroxyalkanoates

A polyhydroxyalkanoate (PHA) synthase gene phaC2 _Ps from Pseudomonas stutzeri strain 1317 was introduced into a PHA synthase gene phbC _Re negative mutant, Ralstonia eutropha PHB⁻4. It conferred on the host strain the ability to synthesize PHA, the monomer compositions of which varied widely when grown on different carbon sources. During cultivation on gluconate, the presence of phaC2 _Ps in R. eutropha PHB⁻4 led to the accumulation of polyhydroxybutyrate (PHB) homopolymer in an amount of 40.9 wt% in dry cells. With fatty acids, the recombinant successfully produced PHA copolyesters containing both short-chain-length and medium-chain-length 3-hydroxyalkanoate (3HA) of 4–12 carbon atoms in length. When cultivated on a mixture of gluconate and fatty acid, the monomer composition of accumulated PHA was greatly affected and the monomer content was easily regulated by the addition of fatty acids in the cultivation medium. After the (R)-3-hydroxydecanol-ACP:CoA transacylase gene phaG _Pp from Pseudomonas putida was introduced into phaC2 _Ps-containing R. eutropha PHB⁻4, poly(3HB-co-3HA) copolyester with a very high 3-hydroxybutyrate (3HB) fraction (97.3 mol%) was produced from gluconate and the monomer compositions of PHA synthesized from fatty acids were also altered. This study clearly demonstrated that PhaC2_Ps cloned from P. stutzeri 1317 has extraordinarily low substrate specificity in vivo, though it has only 54% identity in comparison to a previously described low-substrate-specificity PHA synthase PhaC1_Ps from Pseudomonas sp. 61–3. This study also indicated that the monomer composition and content of the synthesized PHA can be effectively modulated by controlling the addition of carbon sources or by modifying metabolic pathways in the hosts.     

Production of chiral R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters via hydrolytic degradation of polyhydroxyalkanoate synthesized by pseudomonads.

A novel and efficient method for the production of enantiomericaly pure R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters was developed. The described method is based on hydrolysis of poly(hydroxyalkanoate) copolymers synthesized by Pseudomonas putida. The polymer was isolated via solvent recovery and hydrolyzed by acid methanolysis. The obtained 3-hydroxyalkanoic acid methylester mixture was distilled into several fractions with an overall yield of 96.6% (w/w). Gas chromatography-mass spectrometry analysis of the fractions showed that 3-hydroxyhexanoic-, 3-hydroxyoctanoic-, 3 hydroxydecanoic-, and 3-hydroxydodecanoic acid methylesters were enriched to purities exceeding 96 mol%, with distillation yields of 99.9, 99.8, 88.4, and 56.8% (w/w), respectively. Subsequent saponification of the purified methylester fractions yielded the corresponding 3-hydroxyalkanoic acids, which were recovered up to 92.8% (w/w). Chiral gas chromatography analysis confirmed that both 3-hydroxyoctanoic acid and 3-hydroxyoctanoic acid methylester are present in the R-form at a very high enantiomeric excess (>99.9%).     

The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources

We recently identified the phaG(Pp) gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region (herein called phaG(Pa)) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phaG(Pp)-negative mutant P. putida PhaG(N)-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol% 3-hydroxyhexanoate. Furthermore, an isogenic phaG(Pa) knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaG(Pa) mutant did not show any difference in growth rate, but PHA accumulation from gluconate was decreased to about 40% of wild-type level, whereas from fatty acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty acid de novo synthesis towards PHA(MCL) synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.     

Poly-3-hydroxyoctanoate P(3HO), a medium chain length polyhydroxyalkanoate homopolymer from Pseudomonas mendocina

Pseudomonas mendocina was found to produce a unique homopolymer of poly(3-hydroxyoctanoate), P(3HO), rather than a copolymer, when grown on sodium octanoate as the sole carbon source. Although this polymer has been produced by other organisms, interestingly this is the first time an absolute homopolymer has been produced by a wild type organism. In addition, a detailed study on the effects of different extraction methods on the yield, molecular weight, thermal properties, and lipopolysaccharide content of P(3HO) has been carried out. The organism was able to accumulate P(3HO) up to 31.38% of its dry cell weight within 48 h in mineral salt medium. Characterization of the monomer was carried out using FTIR, GC-MS, (13)C, (1)H, and HSQC NMR spectroscopy. The polymer had a crystallinity of 37.5%, Young's modulus value of 11.6 MPa and contact angle of 77.3°. Microstructural studies of solvent cast polymer films revealed a smooth surface topography with a root-mean-square roughness value of 0.238 μm.     

In vivo blending of medium chain length polyhydroxy-alkanoates and polyhydroxybutyrate using recombinant Pseudomonas putida harboring phbCAB operon of Ralstonia eutropha

The in vivo blending of medium chain length polyhydroxyalkanoates (mcl-PHA) and polyhydroxybutyrate (PHB) was carried out using recombinant Pseudomonas putida after transforming the phbCAB operon of Ralstonia eutropha. The most suitable carbon sources for the production of mcl-PHA and PHB blends were identified to be octanoate and gluconate. The molar fractions of 3-hydroxyoctanoate and 3-hydroxybutyrate in the polymer blends were effectively modulated by controlling the mixing ratio of octanoate and gluconate, thereby producing a composition ranging from 95% mcl-PHA to 78% PHB.     

Production of polyhydroxyalkanoates by Pseudomonas nitroreducens

A strain coded AS 1.2343 was isolated from oil-contaminated soil in an oil-field in North China Tianjian City and it was identified as Pseudomonas nitroreducens. The strain demonstrated some unusual ability to synthesize polyhydroxybutyrate (PHB) homopolymer from medium-chain-length (mcl) fatty acids including hexanoate and octanoate. While polyhydroxyalkanoates (PHA) consisting of mcl hydroxyalkanoate (HA) monomers such as hydroxyoctanoate (HO) and hydroxydecanoate (HD) were the major compositions when butyrate, decanoate, lauric acid and tetradecanoic acid were used as substrates for the cell growth, respectively. PHA was accumulated up to 77% of the cell dry weight when growth was conducted in lauric acid, it appeared that the HA contents in the PHA would not be much affected by the changing of the lauric acid concentration. Varying the concentration ratio of butyrate to octanoate could change the composition of PHA accumulated by the strain. Yet PHB homopolymer was always the only polyester synthesized by the strain, regardless of the octanoate concentration change. Additionally, the ratio of carbon to nitrogen (C/N) in butyrate media was found to have effects on the PHA monomer content, as C/N increased from 2 to 100, content of HB decreased from 100% to 7%. PHA polyester synthesized by cells of Pseudomonas nitroreducens AS 1.2343 was a blend polymers consisting of acetone-insoluble HB and acetone-soluble mcl HA monomers.     

Production and characterization of medium-chain-length polyhydroxyalkanoate with high 3-hydroxytetradecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442

Medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate, 3-hydroxydodecanoate, and high-content 3-hydroxytetradecanoate (HTD) was produced by knockout mutant Pseudomonas putida KT2442 termed P. putida KTOY06. When grown on 6 to14 g/L single-carbon-source tetradecanoic acid, P. putida KTOY06, which β-oxidation pathway was weakened by deleting genes of 3-ketoacyl-coenzyme A (CoA) thiolase (fadA) and 3-hydroxyacyl-CoA dehydrogenase (fadB), for the first time, produced several mcl-PHA including 31 to 49 mol% HTD as a major monomer. HHx contents in these mcl-PHAs remained approximately constant at less than 3 mol%. In addition, large amounts of oligo-HTD were detected in cells, indicating the limited ability of P. putida KTOY06 in polymerizing long-chain-length 3-hydroxyalkanoates. The mcl-PHA containing high HTD monomer contents was found to have both higher crystallinity and improved tensile strength compared with that of typical mcl-PHA.     

Enhanced yield of medium-chain-length polyhydroxyalkanoates from nonanoic acid by co-feeding glucose in carbon-limited, fed-batch culture

Medium-chain-length polyhydroxyalkanoates (MCL-PHAs) were produced in carbon-limited, single-stage, fed-batch fermentations of Pseudomonas putida KT2440 by co-feeding nonanoic acid (NA) and glucose (G) to enhance the yield of PHA from NA. An exponential (μ = 0.25 h⁻¹) followed by a linear feeding strategy at a NA:G ratio of 1:1 (w/w) achieved 71 g l⁻¹ biomass containing 56% PHA. Although the same overall PHA productivity (1.44 g l⁻¹ h⁻¹) was obtained when NA alone was fed at the same specific growth rate, the overall yield of PHA from NA increased by 25% (0.66 g PHA g NA⁻¹ versus 0.53 g g⁻¹) with glucose co-feeding. Further increasing glucose in the feed (NA:G = 1:1.5) resulted in a slightly higher yield (0.69 g PHA g NA⁻¹) but lower PHA content (48%) and productivity (1.16 g l⁻¹ h⁻¹). There was very little change in the PHA composition.     

Biochemical observations on medium-chain-length polyhydroxyalkanoate biosynthesis and accumulation in Pseudomonas mendocina

Certain Pseudomonads are capable of accumulating high levels of medium-chain-length polyhydroxyalkanates (PHAmcl) when grown with carbohydrates as the main carbon source. 3-OH acyl components of PHAmcl are derived from fatty acid synthase (FAS) and these components are accessed by action of 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase (transacylase). However, little is known with regard to the time courses of 3-OH acyl component occurrence and of transacylase activity during PHAmcl induction. Also, little is known with regard to the coupling mechanism between FAS and PHAmcl synthesis or whether the FAS pathway itself is specialized in PHAmcl-producing cells. Our results with regard to the time course of formation of 3-OH acids, 3-OH acyl-ACPs, and PHAmcl are consistent with the view that transacylase provides the key link between FAS and PHAmcl synthase. They also suggest that FAS specialization is not a feature of the mechanism. Further, we observed the formation of a 3-OH 10:0 homopolymer early in the induction phase followed by later formation of a mixed polymer containing 3-OH 8:0 and 3-OH 12:0 in addition to 3-OH 10:0. Early occurrence of 3-OH 10:0-CoA transacylase activity was coincident with homopolymer formation.     

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Gluconate and Glucose by Recombinant Aeromonas hydrophila and Pseudomonas putida

Aeromonas hydrophila 4AK4 and Pseudomonas putida GPp104 were genetically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) using gluconate and glucose rather than fatty acids. A truncated tesA gene, encoding cytosolic thioesterase I of Escherichia coli which catalyzes the conversion of acyl-ACP into free fatty acids, was introduced into A. hydrophila 4AK4. When grown in gluconate, the recombinant A. hydrophila 4AK4 synthesized 10% (w/w) PHBHHx containing 14% (mol/mol) 3-hydroxyhexanoate. If additional PHBHHx synthesis genes, phaPCJ, were over-expressed with the truncated tesA in A. hydrophila 4AK4, the PHBHHx content increased to 15% (w/w) and contained 19% (mol/mol) 3-hydroxyhexanoate. Recombinant P. putida GPp104 harboring phaC encoding PHBHHx synthase of A. hydrophila, phaB encoding acetoacetyl-CoA reductase of Wautersia eutropha and phaG encoding 3-hydroxyacyl-ACP-CoA transferase of P. putida, synthesized 19% (w/w) PHBHHx containing 5% (mol/mol) 3-hydroxyhexanoate from glucose. The results suggest that the engineered pathways were applicable to synthesize PHBHHx from unrelated carbon sources such as gluconate and glucose.