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).     

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.     

The role of the fatty acid beta-oxidation multienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: molecular characterization of the fadBA operon from P. oleovorans and of the enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida

In order to investigate the role of the putative epimerase function of the beta-oxidation multienzyme complex (FadBA) in the provision of (R)-3-hydroxyacyl-CoA thioesters for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis, the fadBA(Po) operon of Pseudomonas oleovorans was cloned and characterized. The fadBA(Po) operon and a class-II PHA synthase gene of Pseudomonas aeruginosa were heterologously co-expressed in Escherichia coli to determine whether the putative epimerase function of FadBA(Po) has the ability to provide precursors for PHA accumulation in a non-PHA-accumulating bacterium. Cultivation studies with fatty acids as carbon source revealed that FadBA(Po) did not mediate PHA(MCL) biosynthesis in the E. coli wild-type strain harboring a PHA synthase gene. However, PHA accumulation was strongly impaired in a recombinant E. coli fadB mutant, which harbored a PHA synthase gene. These data indicate that in pseudomonads FadBA does not possess the inherent property, based on a putative epimerase function, to provide the ( R)-enantiomer of 3-hydroxyacyl-CoA efficiently and that other linking enzymes are required to efficiently channel intermediates of beta-oxidation towards PHA(MCL) biosynthesis. However, the phaJ gene from P. oleovorans and from Pseudomonas putida, both of which encoded a 3- Re enoyl-CoA hydratase, was identified. The co-expression of phaJ(Po/Pp) with either a class-II PHA synthase gene or the PHA synthase gene from Aeromonas punctata in E. coli revealed that PhaJ(Po/Pp) mediated biosynthesis of either PHA(MCL), contributing to about 1% of cellular dry mass, or of poly(3-hydroxybutyrate- co-3-hydroxyhexanoate), contributing to 3.6% of cellular dry mass, when grown on decanoate. These data indicate that FadBA(Po)does not mediate the provision of (R)-3-hydroxyacyl-CoA, which resembles FadBA of non-PHA-accumulating bacteria, and that 3- Re enoyl-CoA hydratases are required to divert intermediates of fatty acid beta-oxidation towards PHA biosynthesis in P. oleovorans.     

Heterologous expression of the acyl–acyl carrier protein thioesterase gene from the plant Umbellularia californica mediates polyhydroxyalkanoate biosynthesis in recombinant Escherichia coli

The acyl-acyl carrier protein (ACP) thioesterase cDNA from the plant Umbellularia californica was functionally expressed in various recombinant Escherichia coli strains in order to establish a new metabolic route toward medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis from non-related carbon sources. Coexpression of the PHA synthase genes from Ralstonia eutropha and Pseudomonas aeruginosa, or only the PHA synthase gene from P. aeruginosa, respectively, showed PHA(MCL) accumulation when the type II PHA synthase from P. aeruginosa was produced. Both wild-type E. coli and various fad mutants were investigated; and only when the beta-oxidation pathway was impaired PHA(MCL) accumulation from gluconate was observed, contributing to about 6% of cellular dry weight. Thus coexpression of type II PHA synthase gene with cDNA encoding the medium-chain acyl-ACP thioesterase from U. californica established a new PHA(MCL) biosynthesis pathway, connecting fatty acid de novo biosynthesis with fatty acid beta-oxidation, using a non-related carbon source.     

Swinging effect of salicylic acid on the accumulation of polyhydroxyalkanoic acid (PHA) in Pseudomonas aeruginosa BM114 synthesizing both MCLandSCL-PHA

A bacterium, Pseudomonas aeruginosa BM114, capable of accumulating a blend of medium-chain-length (MCL)- and short-chain-length (SCL)-polyhydroxyalkanoic acid (PHA), was isolated. Salicylic acid (SA), without being metabolized, was found to specifically inhibit only the accumulation of MCL-PHA without affecting cell growth. An addition of 20 mM SA selectively inhibited the accumulation of MCL-PHA in decanoate-grown cells by 83% of the control content in one-step cultivation, where overall PHA accumulation was inhibited by only approximately11%. Typically, the molar monomerunit ratio of the PHA for 25 mM decanoate-grown cells changed from 46:4:25:25 (=[3-hydroxybutyrate]:[3-hydroxycaproate]: [3-hydroxyoctanoate]:[3-hydroxydecanoate]) at 0 mM SA (dry cell wt, 1.97 g/l; PHA content, 48.6 wt%) to 91:1:4:4 at 20 mM SA (dry cell wt, 1.85 g/l; PHA content, 43.2 wt%). Thus, the stimulation of SCL-PHA accumulation was observed. Growth of P. aeruginosa BM114 on undecanoic acid also produced a PHA blend composed of 47.4% P(3HB-co-3- hydroxyvalerate) and 52.6% P(3-hydroxyheptanoate-co-3- hydroxynonanoate-co-3-hydroxyundecanoate). Similar to the case of even-carboxylic acids, SA inhibited the accumulation of only MCL-PHA, but stimulated the accumulation of SCLPHA. For all medium-chain fatty acids tested, SA induced a stimulation of SCL-PHA accumulation in the BM114 strain. SA could thus be used to suppress only the formation of MCL-PHA in Pseudomonas spp. accumulating a blend of SCL-PHA and MCL-PHA.     

Effect of heterologous expression of phaG [(R)-3-hydroxyacyl-ACP-CoA transferase] on polyhydroxyalkanoate accumulation from the aromatic hydrocarbon phenylacetic acid in Pseudomonas species

Five Pseudomonas strains capable of growth with the aromatic carboxylic acid phenylacetic acid were investigated with a view to improving PHA accumulation. The overexpression of (R)-3-hydroxyacyl-ACP-CoA transferase (PhaG) from Pseudomonas putida CA-3 increased PHA accumulation in only one of the five strains tested, namely Pseudomonas jessenii C8. Recombinant P. jessenii C8 harbouring the phaG gene showed a 4.1-fold increase (9.6-39% cell dry weight) in PHA accumulation when grown on phenylacetic acid (15 mM) compared with the wild-type strain. This is the highest reported level of PHA accumulation from phenylacetic acid. This is also the first time the heterologous expression of phaG has resulted in improved PHA accumulation from an aromatic carbon source. The growth patterns of the wild type and recombinant strains were very similar, with no significant differences observed in carbon and nitrogen utilization.     

Accumulation of polyhydroxyalkanoic acid containing large amounts of unsaturated monomers in Pseudomonas fluorescens BM07 utilizing saccharides and its inhibition by 2-bromooctanoic acid.

A psychrotrophic bacterium, Pseudomonas fluorescens BM07, which is able to accumulate polyhydroxyalkanoic acid (PHA) containing large amounts of 3-hydroxy-cis-5-dodecenoate unit up to 35 mol% in the cell from unrelated substrates such as fructose, succinate, etc., was isolated from an activated sludge in a municipal wastewater treatment plant. When it was grown on heptanoic acid (C(7)) to hexadecanoic acid (C(16)) as the sole carbon source, the monomer compositional characteristics of the synthesized PHA were similar to those observed in other fluorescent pseudomonads belonging to rRNA homology group I. However, growth on stearic acid (C(18)) led to no PHA accumulation, but instead free stearic acid was stored in the cell. The existence of the linkage between fatty acid de novo synthesis and PHA synthesis was confirmed by using inhibitors such as acrylic acid and two other compounds, 2-bromooctanoic acid and 4-pentenoic acid, which are known to inhibit beta-oxidation enzymes in animal cells. Acrylic acid completely inhibited PHA synthesis at a concentration of 4 mM in 40 mM octanoate-grown cells, but no inhibition of PHA synthesis occurred in 70 mM fructose-grown cells in the presence of 1 to 5 mM acrylic acid. 2-Bromooctanoic acid and 4-pentenoic acid were found to much inhibit PHA synthesis much more strongly in fructose-grown cells than in octanoate-grown cells over concentrations ranging from 1 to 5 mM. However, 2-bromooctanoic acid and 4-pentenoic acid did not inhibit cell growth at all in the fructose media. Especially, with the cells grown on fructose, 2-bromooctanoic acid exhibited a steep rise in the percent PHA synthesis inhibition over a small range of concentrations below 100 microM, a finding indicative of a very specific inhibition, whereas 4-pentenoic acid showed a broad, featureless concentration dependence, suggesting a rather nonspecific inhibition. The apparent inhibition constant K(i) (the concentration for 50% inhibition of PHA synthesis) for 2-bromooctanoic acid was determined to be 60 microM, assuming a single-site binding of the inhibitor at a specific inhibition site. Thus, it seems likely that a coenzyme A thioester derivative of 2-bromooctanoic acid specifically inhibits an enzyme linking the two pathways, fatty acid de novo synthesis and PHA synthesis. We suggest that 2-bromooctanoic acid can substitute for the far more expensive (2,000 times) and cell-growth-inhibiting PHA synthesis inhibitor, cerulenin.     

Purification of the multienzyme complex for fatty acid oxidation from Pseudomonas fragi and reconstitution of the fatty acid oxidation system

The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3,trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxyacyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240,000. Two types of subunits, with molecular weights of 73,000 and 42,000, were identified. The complex was composed of two copies each of the 73,000- and 42,000-Da subunits. The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.     

Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.     

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 (C₆ to C₁₄) (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 β-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).     

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.     

Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through beta-oxidation

Transgenic plants producing peroxisomal polyhydroxy- alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the beta-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the beta-oxidation of these fatty acids. Tween-laurate was a stronger inducer of beta-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween-oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards beta-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the beta-oxidation cycle as well as removal and degradation of these unusual fatty acids through beta-oxidation. Such enhanced flow of fatty acids through beta-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through beta-oxidation as well as to analyse the degradation pathway of unusual fatty acids.     

Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli

In order to investigate the in vivo substrate specificity of the type I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally expressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid beta-oxidation and the wild-type. The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R. eutropha genes encoding beta-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase comprising the entire PHB operon (pBHR68) as well as in combination with the phaC1 gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase. The fatty acid beta-oxidation route was employed to provide various 3-hydroxyacyl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase. In vivo PHA synthase activity was indicated by PHA accumulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester. Only in recombinant E. coli fad mutants harboring plasmid pBHR68, the R. eutropha PHA synthase led to accumulation of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and decanoate or dodecanoate were provided as carbon source, respectively. Coexpression of phaC1 from P. aeruginosa indicated and confirmed the provision of PHA precursor via the beta-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E. coli strain. These data strongly suggested that R. eutropha PHA synthase accepts, besides the main substrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD.     

Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli.

Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the beta-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.     

Molecular cloning and functional analysis of (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase from Pseudomonas mendocina LZ

An inactive (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase (PhaG(Pm)) was cloned from a newly isolated Proteobacteria Pseudomonas mendocina LZ. It is the first characterized native inactive PhaG protein. Sequence analysis indicated that there were only two sites where the amino acid sequence differed between this inactive protein and the functional PhaG(Pp) from P. putida. The differences were located at position 78 and in the region 109-113 in the amino acid sequence. Mutagenesis was carried out to investigate these two sites. A recombinant strain harboring a S78C PhaG(Pp) mutant accumulated polyhydroxyalkanoates (PHA) at 11.9% of the cellular dry weight, as compared to the 21.6% PHA produced by the recombinant harboring the wild-type PhaG(Pp). On the other hand, the changes in the amino acid region 109-113 of PhaG(Pp) to its corresponding region of PhaG(Pm) resulted in negligible PHA accumulation. This demonstrated that region 109-113 in PhaG is relatively important for transacylase activity, while position 78 just plays a supporting role for the enzyme. Furthermore, 3-D structural models of PhaG(Pp) and PhaG(Pm) developed by computational prediction revealed that the variation in amino acids at 109-113 leads to the destruction of the PhaG catalytic center, resulting in the loss of enzyme activity.     

Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated beta-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and (1)H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of beta-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.     

Biosynthesis of Novel Aromatic Copolyesters from Insoluble 11-Phenoxyundecanoic Acid by Pseudomonas putida BM01

Two types of novel aromatic copolyesters were synthesized from 11-phenoxyundecanoic acid (11-POU) as the sole carbon source and the cosubstrates 11-POU and octanoate, respectively, by isolated Pseudomonas putida BM01 that is known to accumulate high concentrations of medium-chain-length polyesters. Insoluble 11-POU was recrystallized in situ in buffer by alkaline treatment and pH adjustment, followed by autoclaving. The resulting microcrystals, whose structure was different from that of the commercially available crystalline powder, suspended in media were rapidly consumed by the bacterium. Synthesized polymers were characterized by gas chromatography, nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. The aromatic copolyesters synthesized from 11-POU were composed of two monomer units consisting of 3-hydroxy-5-phenoxyvalerate (5POHV) as the major component (72 to 85 mol%) and 3-hydroxy-7-phenoxyheptanoate (7POHH) as the minor component (15 to 28 mol%). The aromatic copolyesters showed a crystalline melting transition at 70(deg)C. When the bacterium was grown on the cosubstrates 11-POU and octanoate, the bacterium synthesized the copolyesters composed of aromatic and aliphatic monomers poly(5POHV-co-7POHH-co-3-hydroxy-9-phenoxynonanoate-co-3-hydroxyalkanoates) . The addition of octanoate in the feed shifted the major monomer unit in the polymer from 5POHV to 7POHH. A further-fragmented metabolite, 3-phenoxypropionate, whose concentration reached a steady state at the time of greatest polyester accumulation, was detected in the medium. The metabolic pathway of 11-POU is suggested.