Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids

Burkholderia sp. accumulates polyhydroxyalkanoates (PHAs) containing 3-hydroxybutyrate and 3-hydroxy-4-pentenoic acid when grown on mineral media under limited phosphate or nitrogen, and using sucrose or gluconate as a carbon and energy source. Solvent fractionation and NMR spectroscopic characterization of these polyesters revealed the simultaneous accumulation of two homopolyesters rather than a co-polyester with random sequence distribution of the monomers [Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbüchel A, Tran M, Asrar J (1999) Macromolecules 32: 7389–7395]. To understand the genetic requirements for such unusual polyester accumulation, we probed total genomic DNA from Burkholderia sp. by Southern hybridization experiments using phaC-specific probes. These experiments indicated the presence of more than one PHA synthase gene within the genome of Burkholderia sp. However, when total genomic DNA from Burkholderia sp. was used to complement a PHA-negative mutant of Ralstonia eutropha for PHA accumulation, only one PHA synthase gene was obtained resembling the R. eutropha type of PHA synthases, based on amino acid sequence similarity. In addition to the PHA synthase gene, based on high sequence homology, genes encoding a β-ketothiolase and acetoacetyl-CoA reductase were identified in a gene cluster with the PHA synthase gene. The arrangement of the three genes is quite similar to the R. eutropha poly-β-hydroxybutyrate biosynthesis operon. Received: 3 September 1999 / Received revision: 29 October 1999 / Accepted: 5 November 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.     

Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalkanoate Synthase (phaC) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109

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

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome‐targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β‐oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.     

In vivo evolution of the Aeromonas punctata polyhydroxyalkanoate (PHA) synthase: isolation and characterization of modified PHA synthases with enhanced activity

In vivo random mutagenesis of the polyhydroxyalkanoate (PHA) synthase gene from Aeromonas punctata was performed employing the mutator strain Escherichia coli XL1-Red. About 200,000 mutants were screened on Nile red-containing medium and five mutants with enhanced fluorescence were selected. Four of these mutants exhibited enhanced in vivo and in vitro PHA synthase activity. Mutant M1, which carried the single mutation F518I, showed a five-fold increase in specific PHA synthase activity, whereas the corresponding mediated PHA accumulation increased by 20%, as compared with the wild-type PHA synthase. Mutant M2, which carried the single mutation V214G, showed a two-fold increase in specific PHA synthase activity and PHA accumulation only increased by 7%. Overall, the in vitro activities of the overproducing mutants ranged from 1.1- to 5-fold more than the wild-type activity, whereas the amounts of accumulated PHA ranged over 107–126% of that of the wild type. Moreover, all mutants mediated synthesis of PHAs with an increased weight average molar mass, but the molar fractions of 3-hydroxybutyrate and 3-hydroxyhexanoate remained almost constant. In vivo random mutagenesis proved to be a versatile tool to isolate mutants exerting improved properties with respect to PHA biosynthesis. Electronic Publication     

Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects

Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.     

Rapid genetic characterization of poly(hydroxyalkanoate) synthase and its applications

Microorganisms containing short-chain-length (scl-) or medium-chain-length (mcl-) poly(hydroxyalkanoates) (PHAs) are commonly screened by applying rapid staining methods using lipophilic reagents. These methods provide powerful means for general screening of organisms actively producing and accumulating PHAs. The Southern blot hybridization method additionally allows the identification of potential PHA-producing microorganisms. Polymerase chain reaction (PCR)-based detection methods further afford rapid and sensitive means to screen for PHA biosynthesis genes. Specific PCR assays had been developed for the simultaneous or individual detection of the class II mcl-PHA synthase genes of Pseudomonas. The amplicons (approximately 0.54 kb) can be directly sequenced or used as probes for hybridization studies. The sequence information can further be used to initiate chromosome walking for an eventual cloning of the complete PHA biosynthesis operon. In addition, the amplification pattern and sequence data can be used to differentiate subgroups of organisms, as demonstrated for P. corrugata and P. mediterranea. Other researchers reported PCR methods for the detection of scl-PHA synthase genes and those of Bacillus spp., thus greatly expanding the types of PHA synthase gene and the organisms that can be characterized by this approach. The vast sequence information obtainable through PCR-based studies of various PHA synthase operons should facilitate the identification or construction of new PHA synthases capable of synthesizing novel PHAs.     

Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Bacillus subtilis as potential producer for polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.     

Biosynthetic polyesters consisting of 2-hydroxyalkanoic acids: current challenges and unresolved questions

2-Hydroxyalkanoates (2HAs) have become the new monomeric constituents of bacterial polyhydroxyalkanoates (PHAs). PHAs containing 2HA monomers, lactate (LA), glycolate (GL), and 2-hydroxybutyrate (2HB) can be synthesized by engineered microbes in which the broad substrate specificities of PHA synthase and propionyl-CoA transferase are critical factors for the incorporation of the monomers into the polymer chain. LA-based polymers, such as P[LA-co-3-hydroxybutyrate (3HB)], have the properties of pliability and stretchiness which are distinctly different from those of the rigid poly(lactic acid) (PLA) and P(3HB) homopolymers. This versatile platform is also applicable to the biosynthesis of GL- and 2HB-based polymers. In the case of the synthesis of 2HB-based polymers, the enantiospecificity of PHA synthase enabled the production of isotactic (R)-2HB-based polymers, including P[(R)-2HB], from racemic precursors of 2HB. P(2HB) is a pliable material, in contrast to PLA. Furthermore, to obtain a new 2HA-polymerizing PHA synthase, the class I PHA synthase from Ralstonia eutropha was engineered so as to achieve the first incorporation of LA units. The analysis of the polymer synthesized using this new LA-polymerizing PHA synthase unexpectedly focused a spotlight on the studies on block copolymer biosynthesis.     

Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (M_w: 480,000 g.mol^–1) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (M_w: 391,000 g mol^–1) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a -ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a K_m of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70°C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 M CoA and lost all of its activity when it was treated with alkaline phosphatase. PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.     

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L^?1 h^?1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.     

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     

Inactivation of type I polyhydroxyalkanoate synthase in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> resulted in discovery of another potential PHA synthase

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase (PhaC) produced only PHBHHx from lauric acid but not from glucose. Medium-chain-length (mcl) PHA was produced from lauric acid or glucose only when PhaC of A. hydrophila was inactivated, indicating the existence of another PHA synthase in the wild type. Using PCR cloning strategy, the potential PHA synthase gene (phaC _mcl) was obtained from genomic DNA of the wild type and exhibited strong homology to type II PHA synthase genes of Pseudomonas strains. The phaC _mcl gene was PCR subcloned into plasmid pBBR1MCS2 and expressed in a PHA-negative mutant of Pseudomonas putida. Recombinant P. putida synthesized mcl PHA from gluconate or octanoate. This result proved that wild type A. hydrophila possessed another type II PHA synthase, which was responsible for the synthesis of mcl PHA, besides type I PHA synthase.     

Expression of 3-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Genes Enhances Production of Polyhydroxyalkanoate Copolymer from Glucose in Recombinant Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.     

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     

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

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

Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 10⁵, and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T _g, T _m, and T _d (10%) were observed at −1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.     

Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.     

Identification and Analysis of the Polyhydroxyalkanoate-Specific β-Ketothiolase and Acetoacetyl Coenzyme A Reductase Genes in the Cyanobacterium Synechocystis sp. Strain PCC6803

Synechocystis sp. strain PCC6803 possesses a polyhydroxyalkanoate (PHA)-specific β-ketothiolase encoded by phaA_Syn and an acetoacetyl-coenzyme A (CoA) reductase encoded by phaB_Syn. A similarity search of the entire Synechocystis genome sequence identified a cluster of two putative open reading frames (ORFs) for these genes, slr1993 and slr1994. Sequence analysis showed that the ORFs encode proteins having 409 and 240 amino acids, respectively. The two ORFs are colinear and most probably coexpressed, as revealed by sequence analysis of the promoter regions. Heterologous transformation of Escherichia coli with the two genes and the PHA synthase of Synechocystis resulted in accumulation of PHAs that accounted for up to 12.3% of the cell dry weight under high-glucose growth conditions. Targeted disruption of the above gene cluster in Synechocystis eliminated the accumulation of PHAs. ORFs slr1993 and slr1994 thus encode the PHA-specific β-ketothiolase and acetoacetyl-CoA reductase of Synechocystis and, together with the recently characterized PHA synthase genes in this organism (S. Hein, H. Tran, and A. Steinbüchel, Arch. Microbiol. 170:162–170, 1998), form the first complete PHA biosynthesis pathway known in cyanobacteria. Sequence alignment of all known short-chain-length PHA-specific acetoacetyl-CoA reductases also suggests an extended signature sequence, VTGXXXGIG, for this group of proteins. Phylogenetic analysis further places the origin of phaA_Syn and phaB_Syn in the γ subdivision of the division Proteobacteria.