Poly(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(β-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760–12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a β-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(β-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation. Received: 26 May 1999 / Accepted: 16 June 1999     

Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase. Received: 16 March 1999 / Received revision: 24 August 1999 / Accepted: 24 September 1999     

Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production

Screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth. Ralstonia eutropha H16 and Pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (PHA). R. eutropha and P. oleovorans accumulated PHA amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in mineral salt medium with the oil from the rhamnose production as the sole carbon source. The accumulated PHA isolated from R. eutropha consisted of only 3-hydroxybutyric acid, whereas the PHA isolated from P. oleovorans consisted of 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxy decanoic acid, and 3-hydroxydodecanoic acid. The composition was confirmed by gas chromatography of the isolated polyesters. Batch and fed-batch cultivations in stirred-tank reactors were done. Received: 15 June 1999 / Received revision: 10 August 1999 / Accepted: 13 August 1999     

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.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbA_Re) and NADPH-dependent acetoacetyl-CoA reductase (PhbB_Re) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1 _Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB⁻4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1 _Ps gene only. In addition, recombinant strains of R. eutropha PHB⁻4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB⁻4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units. Received: 12 July 1999 / Received revision: 1 October 1999 / Accepted: 2 October 1999     

Production of P(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) Terpolymers Using a Chimeric PHA Synthase in Recombinant Ralstonia eutropha and Pseudomonas putida

Recombinant strains of Ralstonia eutropha and Pseudomonas putida harboring a chimeric polyhydroxyalkanoate (PHA) synthase, which consisted of PHA synthases of Aeromonas caviae and R. eutropha, produced 3-hydroxybutyrate (3HB)-based PHA copolymers comprised of 3-hydroxyhexanoate and 3-hydroxyoctanoate units from dodecanoate (87–97 mol % 3HB), indicating that the chimeric PHA synthase possesses desirable substrate specificity leading to the production of 3HB-rich copolymers.     

Formation of new polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate monomer in Burkholderia sp

Burkholderia sp. synthase has been shown to polymerize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate, and 3-hydroxy-4-pentenoic acid monomers. This study was carried out to evaluate the ability of Burkholderia sp. USM (JCM 15050) and its transformant harboring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae to incorporate the newly reported 3-hydroxy-4-methylvalerate (3H4MV) monomer. Various culture parameters such as concentrations of nutrient rich medium, fructose and 4-methylvaleric acid as well as harvesting time were manipulated to produce P(3HB-co-3H4MV) with different 3H4MV compositions. The structural properties of PHA containing 3H4MV monomer were investigated by using nuclear magnetic resonance and Fourier transform infrared spectroscopy (FTIR). The relative intensities of the bands at 1,183 and 1,228 cm⁻¹ in the FTIR spectra enabled the rapid detection and differentiation of P(3HB-co-3H4MV) from other types of PHA. In addition, the presence of 3H4MV units in the copolymer was found to considerably lower the melting temperature and enthalpy of fusion values compared with poly(3-hydroxybutyrate) (P(3HB)). The copolymer exhibited higher thermo-degradation temperature but similar molecular weight and polydispersity compared with P(3HB).     

Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids)

A gene transfer system for Rhodococcus opacus PD630 based on electroporation was established and optimized employing the Escherichia coli-Rhodococcus shuttle vectors pNC9501 and pNC9503 as well as the E. coli-Corynebacterium glutamicum shuttle vector pJC1 as suitable cloning vectors for R. opacus PD630, resulting in transformation efficiencies up to 1.5 × 10⁵ CFUs/μg plasmid DNA. Applying the optimized electroporation protocol to the pNC9501-derivatives pAK68 and pAK71 harboring the entire PHB synthesis operon from Ralstonia eutropha and the PHA synthase gene phaC1 from Pseudomonas aeruginosa, respectively, recombinant PHA biosynthesis was established in R. opacus PD630 and the TAG-negative mutant ROM34. Plasmid pAK68 enabled synthesis and accumulation of poly(3HB) in R. opacus PD630 and ROM34 during cultivation under storage conditions from 1% (w/v) gluconate, of poly(3HB-co-3HV) from 0.2% (w/v) propionate and of poly(3HV) from 0.1% (w/v) valerate. Under storage conditions, recombinant strains of PD630 and ROM34 harboring pAK71 were able to synthesize and accumulate PHA of the medium chain length hydroxyalkanoic acids 3HHx, 3HO, 3HD and 3HDD from 0.1% (w/v) hexadecane or octadecane and a copolyester composed of 3HHp, 3HN and 3HUD from 0.1% (w/v) pentadecane or heptadecane. In the recombinant strains of PD630 and ROM34, the thiostrepton-induced overexpression of a 20 kDa protein was observed with its N-terminus exhibiting a homology of 60% identical amino acids to TipA from Streptomyces lividans. Received: 13 March 1999 / Received revision: 18 May 1999 / Accepted: 21 May 1999     

Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB^–4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction. Received: 11 March 1998 / Accepted: 2 June 1998     

Physiological–biochemical properties and the ability to synthesize polyhydroxyalkanoates of the glucose-utilizing strain of the hydrogen bacterium Ralstonia eutropha B8562

Physiological–biochemical, genetic, and cultural properties of the glucose-utilizing mutant strain Ralstonia eutropha B8562 have been compared with those of its parent strain R. eutropha B5786. It has been shown that growth characteristics of the strain cultured on glucose as the sole carbon and energy source are comparable with those of the parent strain. Strain B8562 is characterized by high polyhydroxyalkanoate (PHA) yields on different carbon sources (CO₂, fructose, and glucose). PHA accumulation in the strain batch cultured on glucose under nitrogen deficiency reaches 90 %. The major monomer in the PHA is β-hydroxybutyric acid (more than 99 mol %); the identified minor components are β-hydroxyvaleric acid (0.25–0.72 mol %) and β-hydroxyhexanoic acid (0.08–1.5 mol %). The strain is a promising PHA producer on available sugar-containing media with glucose.     

Cloning and functional expression of the d-β-hydroxybutyrate dehydrogenase gene of Rhodobacter sp. DSMZ 12077

Nucleotide sequence and biochemical analysis of d-β-hydroxybutyrate dehydrogenase (EC 1.1.1.30), isolated from Rhodobacter sp., indicate functional oligomers composed of subunits of 257 amino acids with a calculated M _r of 26,800 and a pI of 5.90. Compared to mammalian short-chain alcohol dehydrogenases, the bacterial enzyme lacks a C-terminal lipid anchor domain and was found to be highly active upon expression in Escherichia coli even without lipid supplement. The recombinant enzyme could be highly enriched using a single chromatography step and was shown to be stable over a broad range of pH and temperature. Received: 1 April 1999 / Received last revision: 11 June 1999 / Accepted: 11 June 1999     

Cloning and molecular organization of the polyhydroxyalkanoic acid synthase gene (phaC) of Ralstonia eutropha strain B5786

Class I polyhydroxyalkanoic acid (PHA) synthase gene (phaC) of Ralstonia eutropha strain B5786 was cloned and characterized. R. eutropha B5786 features the ability to synthesize multicomponent PHAs with short- and medium-chain-length monomers from simple carbohydrate substrate. A correlation was made between the molecular structure of PHA synthase and substrate specificity and the ability of strain-producers to accumulate PHAs of this or that structure. A strong similarity of PHA synthase of R. eutropha strain B5786 with PHA synthase of R. eutropha strain H16, which, as opposed to strain B5786, enables to incorporate medium chain length PHAs if hexanoate is used as carbon source, exhibited 99%. A correlation between the structure of PHA synthase of B5786 strain with synthases of microorganisms which synthesize short and medium chain length PHAs similarly to B5786 strain, showed an identity level from 26 to 41% (homology with synthase of Rhodospirillum rubrum makes 41%, Ectothiorhodospira shaposhnikovii makes 26%, Aeromonas punctata makes 40%, Thiococcus pfennigii makes 28%, Rhodococcus ruber makes 38%, and with PhaCl and PhaC2 synthases of Pseudomonas sp. 61–3 makes 34 and 37%, respectively). This allows for speaking about the absence of a direct connection between the molecular organization of PHA synthases and their functional abilities, namely, the ability to synthesize PHAs of a particular composition.     

Physiological and Biochemical Characteristics and Capacity for Polyhydroxyalkanoates Synthesis in a Glucose-Utilizing Strain of Hydrogen-Oxidizing Bacteria, Ralstonia eutropha B8562

The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO₂, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, β-hydroxybutyrate was predominant (over 99 mol %); β-hydroxyvalerate (0.25–0.72 mol %) and β-hydroxyhexanoate (0.008–1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.     

Biosynthesis of polyhydroxyalkanoate homopolymers by Pseudomonas putida

Pseudomonas putida KT2442 has been a well-studied producer of medium-chain-length (mcl) polyhydroxyalkanoate (PHA) copolymers containing C6 ~ C14 monomer units. A mutant was constructed from P. putida KT2442 by deleting its phaG gene encoding R-3-hydroxyacyl-ACP-CoA transacylase and several other β-oxidation related genes including fadB, fadA, fadB2x, and fadAx. This mutant termed P. putida KTHH03 synthesized mcl homopolymers including poly(3-hydroxyhexanoate) (PHHx) and poly(3-hydroxyheptanoate) (PHHp), together with a near homopolymer poly(3-hydroxyoctanoate-co-2 mol% 3-hydroxyhexanoate) (PHO*) in presence of hexanoate, heptanoate, and octanoate, respectively. When deleted with its mcl PHA synthase genes phaC1 and phaC2, the recombinant mutant termed P. putida KTHH08 harboring pZWJ4-31 containing PHA synthesis operon phaPCJ from Aeromonas hydrophila 4AK4 accumulated homopolymer poly(3-hydroxyvalerate) (PHV) when valerate was used as carbon source. The phaC deleted recombinant mutant termed P. putida KTHH06 harboring pBHH01 holding PHA synthase PhbC from Ralstonia eutropha produced homopolymers poly(3-hydroxybutyrate) (PHB) and poly(4-hydroxybutyrate) using γ-butyrolactone was added as precursor. All the homopolymers were physically characterized. Their weight average molecular weights ranged from 1.8 × 10⁵ to 1.6 × 10⁶, their thermal stability changed with side chain lengths. The derivatives of P. putida KT2442 have been developed into a platform for production of various PHA homopolymers.     

Non-conventional yeasts as producers of polyhydroxyalkanoates—genetic engineering of<Emphasis Type="Italic"> Arxula adeninivorans</Emphasis>

The non-conventional yeast Arxula adeninivorans was equipped with the genes phbA, phbB and phbC of the polyhydroxyalkanoate (PHA) biosynthetic pathway of Ralstonia eutropha, which encode -ketothiolase, NADPH-linked acetoacetyl-CoA reductase and PHA synthase, respectively. Arxula strains transformed solely with the PHA synthase gene (phbC) were able to produce PHA. However, the maximum content of the polymer detected in these strains was just 0.003% poly-3-hydroxybutyrate (PHB) and 0.112% poly-3-hydroxyvalerate (PHV). The expression of all three genes (phbA, phbB, phbC) resulted in small increases in the PHA content of the transgenic Arxula cells. However, under controlled cultivation conditions with minimal medium and ethanol as the carbon source, the recombinant yeast was able to accumulate up to 2.2% PHV and 0.019% PHB. Possible reasons for these differences are discussed.     

Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain

The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB⁻4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4–5 mol% whatever the structure of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200–400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains. Received: 3 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa

For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa applying N-terminal His₆-tag fusions and metal chelate affinity chromatography. In vivo His₆-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes. The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg⁻¹ and 0.035 U mg⁻¹ protein, respectively. Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hydroxydecanoyl-CoA as substrate. Specific enzyme activity was increased to 0.055 U mg⁻¹ when the phasin GA24 from Ralstonia eutropha was added to the assay. CoA inhibited PHA synthase activity, and a K _i of 85 μM was determined. A two-enzyme system was established, employing commercially available acyl-CoA synthetase and PHA synthase, which allowed the in vitro de novo PHA granule formation and the in vitro synthesis of poly(3-hydroxydecanoate) exhibiting a weight average molar mass of 9.8 × 10⁴ g mol⁻¹, and which occurred independently of pre-existing PHA granules. Received: 3 December 1999 / Revision received: 10 January 2000 / Accepted: 14 January 2000     

Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126

The time course of the accumulation of triacylglycerols (TAGs) in Rhodococcus opacus PD630 or of TAGs plus polyhydroxyalkanoates (PHA) in Rhodococcus ruber NCIMB 40126 with gluconate or glucose as carbon source, respectively, was studied. In addition, we examined the mobilization of these storage compounds in the absence of a carbon source. R. opacus accumulated TAGs only after the exhaustion of ammonium in the medium, and, with a fixed concentration of the carbon source, the amounts of TAGs in the cells increased with decreasing concentrations of ammonium in the medium. When these cells were incubated in the absence of an additional carbon source, about 90% of these TAGs were mobilized and used as endogenous carbon source, particularly if ammonium was available. R. ruber accumulated a copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate already during the early exponential growth phase, whereas TAGs were synthesized and accumulated mainly during the late exponential and stationary growth phases. In the stationary growth phase, synthesis of TAGs continued, whereas PHA was partially mobilized. In the absence of an additional carbon source but in the presence of ammonium, mobilization of TAGs started first and was then paralleled by the mobilization of PHA, resulting in an approximately 90% and 80% decrease of these storage compounds, respectively. During the accumulation phase, interesting shifts in the composition of the two storage compounds occurred, indicating that the substrates of the PHA synthase and the TAG synthesizing enzymes were provided to varying extents, depending on whether the cells were in the early or late exponential or in the stationary growth phase. Received: 12 January 2000 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme

The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (M _r 40,950) and PhaC (M _r 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed. Received: 9 January 2000 / Received revision: 20 February 2000 / Accepted: 25 February 2000     

Altered composition of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> poly(hydroxyalkanoate) through expression of PHA synthase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> ATCC 35206

The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (<66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved α/β-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a poly(hydroxybutanoate) (PHB)-negative mutant of Ralstonia eutropha PHB⁻4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHB, albeit at a low level (4–5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.