The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.     

New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate

Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.     

Studies on the influence of phasins on accumulation and degradation of PHB and nanostructure of PHB granules in ralstonia eutropha H16

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [PHB] granules and affect their size and number in the cells. Recent studies on the PHB granule proteome and analysis of the complete genomic DNA sequence of Ralstonia eutropha H16 have identified three homologues of the phasin protein PhaP1. In this study, mutants of R. eutropha deficient in the expression of the phasin genes phaP1, phaP2, phaP3, phaP4, phaP12, phaP123, and phaP1234 were examined by gas chromatography. In addition, the nanostructures of the PHB granules of the wild-type and of the mutants were imaged by atomic force microscopy (AFM), and the molecular masses of the accumulated PHB were analyzed by gel permeation chromatography. For this, cells were cultivated under conditions permissive for accumulation of PHB and were then cultivated under conditions permissive for degradation of PHB. Mutants deficient in the expression of phaP2, phaP3, or phaP4 genes mobilized the stored PHB only slowly like the wild-type, whereas degradation occurred much earlier and faster in the phaP1 single mutant as well as in all multiple mutants defective in the phaP1 gene plus one or more other phasin genes. This indicated that the presence of the major phasin PhaP1 on the granule surface is important for PHB degradation and that this phasin is therefore of particular relevance for PHB accumulation. It was also shown that the molecular weights of the accumulated PHB were identical in all examined strains; phasins have therefore no influence on the molecular weight of PHB. The AFM images obtained in this study showed that the PHB granules of R. eutropha H16 form a single interconnected system inside the wild-type cells.     

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins,PhaP6 and PhaP7, in Ralstonia eutropha H16

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with “phasin 2 motifs.” To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.     

Identification of the haloarchaeal phasin (PhaP) that functions in polyhydroxyalkanoate accumulation and granule formation in Haloferax mediterranei

The polyhydroxyalkanoate (PHA) granule-associated proteins (PGAPs) are important for PHA synthesis and granule formation, but currently little is known about the haloarchaeal PGAPs. This study focused on the identification and functional analysis of the PGAPs in the haloarchaeon Haloferax mediterranei. These PGAPs were visualized with two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). The most abundant protein on the granules was identified as a hypothetical protein, designated PhaP. A genome-wide analysis revealed that the phaP gene is located upstream of the previously identified phaEC genes. Through an integrative approach of gene knockout/complementation and fermentation analyses, we demonstrated that this PhaP is involved in PHA accumulation. The ΔphaP mutant was defective in both PHA biosynthesis and cell growth compared to the wild-type strain. Additionally, transmission electron microscopy results indicated that the number of PHA granules in the ΔphaP mutant cells was significantly lower, and in most of the ΔphaP cells only a single large granule was observed. These results demonstrated that the H. mediterranei PhaP was the predominant structure protein (phasin) on the PHA granules involved in PHA accumulation and granule formation. In addition, BLASTp and phylogenetic results indicate that this type of PhaP is exclusively conserved in haloarchaea, implying that it is a representative of the haloarchaeal type PHA phasin.     

Biosynthesis of polyhydroxyalkanoates (PHA) by recombinant Ralstonia eutropha and effects of PHA synthase activity on in vivo PHA biosynthesis.

Recombinant strains of Ralstonia eutropha PHB 4, which harbored Aeromonas caviae polyhydroxyalkanoates (PHA) biosynthesis genes under the control of a promoter for R. eutropha phb operon, were examined for PHA production from various alkanoic acids. The recombinants produced poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from hexanoate and octanoate, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxypentano ate) [P(3HB-co-3HV-co-3HHp)] from pentanoate and nonanoate. One of the recombinant strains, R. eutropha PHB 4/pJRDBB39d3 harboring ORF1 and PHA synthase gene of A. caviae (phaC(Ac)) accumulated copolyesters with much more 3HHx or 3HHp fraction than the other recombinant strains. To investigate the relationship between PHA synthase activity and in vivo PHA biosynthesis in R. eutropha, the PHB- 4 strains harboring pJRDBB39d13 or pJRDEE32d13 were used, in which the heterologous expression of phaC(Ac) was controlled by promoters for R. eutropha phb operon and A. caviae pha operon, respectively. The PHA contents and PHA accumulation rates were similar between the two recombinant strains in spite of the quite different levels of PHA synthase activity, indicating that the polymerization step is not the rate-determining one in PHA biosynthesis by R. eutropha. The molecular weights of poly(3-hydroxybutyrate) produced by the recombinant strains were also independent of the levels of PHA synthase activity. It has been suggested that a chain-transfer agent is generated in R. eutopha cells to regulate the chain length of polymers.     

Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.     

Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.     

Engineered Aeromonas hydrophila for enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with alterable monomers composition

Aeromonas hydrophila 4AK4 produces poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 3-hydroxybutyrate (3HB) and about 15 mol% 3-hydroxyhexanoate (3HHx) from dodecanoate. To study the factors affecting the monomer composition and PHBHHx content, genes encoding phasin (phaP), PHA synthase (phaC) and (R)-specific enoyl-CoA hydratase (phaJ) from Aeromonas punctata (formerly named Aeromonas caviae) were introduced individually or jointly into A. hydrophila 4AK4. The phaC gene increased 3HHx fraction more significantly than phaP, while phaJ had little effect. Expression of phaC alone increased the 3HHx fraction from 14 to 22 mol%. When phaC was co-expressed with phaP and phaJ, the 3HHx fraction increased from 14 to 34 mol%. Expression of phaP or phaC alone or with another gene enhanced PHBHHx content up to 64%, cell dry weight (CDW) as much as 4.4 gL(-1) and PHBHHx concentration to 2.7 gL(-1) after 48 h in shake flask culture. The results suggest that a higher PHA synthase activity could lead to a higher 3HHx fraction and PHBHHx content. Co-expression of phaJ with phaC or phaP would favor PHA accumulation, although over-expression of phaJ did not affect PHA synthesis much. In addition, inhibition of beta-oxidation by acrylate in A. hydrophila 4AK4 enhanced PHBHHx content. However, no monomers longer than 3HHx were detected. The results show that genetic modification of A. hydrophila 4AK4 enhanced PHBHHx production and altered monomer composition of the polymer.     

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.     

Formation of short chain length/medium chain length polyhydroxyalkanoate copolymers by fatty acid beta-oxidation inhibited Ralstonia eutropha

Ralstonia eutropha has been considered as a bacterium, incorporating hydroxyalkanoates of less than six carbons only into polyhydroxyalkanoates (PHAs). Cells of the wild type cultivated with sodium octanoate as the carbon source in the presence of the fatty acid beta-oxidation inhibitor sodium acrylate synthesized PHAs composed of the medium chain length hydroxyalkanoates (3HA(MCL)) 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate (3HO) as well as of 3-hydroxybutyrate and 3-hydroxyproprionate as revealed by gas chromatography, (1)H NMR spectroscopy, and mass spectroscopy. The characterization of the polymer as a tetrapolymer was confirmed by differential solvent extraction and measurement of melting and glass transition temperature depression in the purified polymer compared to PHB. These data suggested that the R. eutropha PHA synthase is capable of incorporating longer chain substrates than suggested by previous in vitro studies. Furthermore, expression of the class II PHA synthase gene phaC1 from P. aeruginosa in R. eutropha resulted in the accumulation of PHAs consisting of 3HA(MCL) contributing about 3-5% to cellular dry weight. These PHAs were composed of nearly equal molar fractions of 3HO and 3-hydroxydecanoate (3HD) with traces of 3HHx. These data indicated that 3HA(MCL)-CoA thioesters were diverted from the fatty acid beta-oxidation pathway towards PHA biosynthesis in recombinant R. eutropha.     

Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases PhaC1 and PhaC2

This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P mendocina pha gene locus, encoding two PHA synthase genes [phaC1Pm and phaC2pm flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2pm (1,680 bp) were determined. The amino acid sequences deduced from phaC1Pm and phaC2pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P. putida GPp104 and Ralstonia eutropha PHB-4. In P. putida GPp 104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6-C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB-4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA. Recombinant strains of R. eutropha PHB-4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P. mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.     

Binding of the major phasin, PhaP1, from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1(Reu) is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1(Reu) which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1(Reu) or various regions of PhaP1(Reu) were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1(Reu) contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1(Reu), which revealed that PhaP1(Reu) is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.     

Engineering of chimeric class II polyhydroxyalkanoate synthases

PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.     

<Emphasis Type="Italic">Burkholderia xenovorans</Emphasis> LB400 possesses a functional polyhydroxyalkanoate anabolic pathway encoded by the <Emphasis Type="Italic">pha</Emphasis> genes and synthesizes poly(3-hydroxybutyrate) under nitrogen-limiting conditions

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that are synthesized by diverse bacteria. In this study, the synthesis of PHAs by the model aromatic-degrading strain Burkholderia xenovorans LB400 was analyzed. Twelve pha genes including three copies of phaC and five copies of the phasin-coding phaP genes are distributed among the three LB400 replicons. The phaC1ABR gene cluster that encodes the enzymes of the PHA anabolic pathway is located at chromosome 1 of strain LB400. During the growth of strain LB400 on glucose under nitrogen limitation, the expression of the phaC1, phaA, phaP1, phaR, and phaZ genes was induced. Under nitrogen limitation, PHA accumulation in LB400 cells was observed by fluorescence microscopy after Nile Red staining. GC-MS analyses revealed that the PHA accumulated under nitrogen limitation was poly(3-hydroxybutyrate) (PHB). LB400 cells grown on glucose as the sole carbon source under nitrogen limitation accumulated 40?±?0.96% PHB of the cell dry weight, whereas no PHA was observed in cells grown in control medium. The functionality of the phaC1 gene from strain LB400 was further studied using heterologous expression in a Pseudomonas putida KT40C1ZC2 mutant strain derived from P. putida KT2440 that is unable to synthesize PHAs. Interestingly, KT40C1ZC2[pVNC1] cells that express the phaC1 gene from strain LB400 were able to synthesize PHB (33.5% dry weight). This study indicates that B. xenovorans LB400 possesses a functional PHA synthetic pathway that is encoded by the pha genes and is capable of synthesizing PHB.     

Polyhydroxyalkanoate (PHA) accumulation in sulfate-reducing bacteria and identification of a class III PHA synthase (PhaEC) in Desulfococcus multivorans

Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs). During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)]. Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter. Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate. Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected. When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained. The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaE(Dm) and phaC(Dm) genes. PhaC(Dm) and PhaE(Dm) were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases. Constructs of phaC(Dm) alone (pBBRMCS-2::phaC(Dm)) and of phaE(Dm)C(Dm) (pBBRMCS-2::phaE(Dm)C(Dm)) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB(-)4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaE(Dm)C(Dm) small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected. This indicated that the cloned genes encode functionally active proteins. Hybrid synthases consisting of PhaC(Dm) and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 were also constructed and were shown to be functionally active.     

Effective enhancement of short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production by coexpression of genetically engineered 3-ketoacyl-acyl-carrier-protein synthase III (fabH) and polyhydroxyalkanoate synthesis genes

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that have a wide variety of physical properties dependent on the lengths of the pendant groups of the monomer units in the polymer. PHAs composed of mostly short-chain-length (SCL) monomers are often stiff and brittle, whereas PHAs composed of mostly medium-chain-length (MCL) monomers are elastomeric in nature. SCL-MCL PHA copolymers can have properties between the two states, dependent on the ratio of SCL and MCL monomers in the copolymer. It is desirable to elucidate new and low cost ways to produce PHA composed of mostly SCL monomer units with a small mol % of MCL monomers from renewable resources, since this type of SCL-MCL PHA copolymer has superior qualities compared to SCL homopolymer. To address this issue, we have created strains of recombinant E. coli capable of producing beta-ketothiolase (PhbA) and acetoacetyl-CoA synthase (PhbB) from Ralstonia eutropha, genetically engineered 3-ketoacyl-ACP synthase III (FabH) from Escherichia coli, and genetically engineered PHA synthases (PhaC) from Pseudomonas sp. 61-3 to enhance the production of SCL-MCL PHA copolymers from glucose. The cumulative effect of having two monomer-supplying pathways and genetically engineered PHA synthases resulted in higher accumulated amounts of SCL-MCL PHA copolymer from glucose. Polymers were isolated from two recombinant E. coli strains, the first harboring the phbAB, fabH(F87T), and phaC1(SCQM) genes and the second harboring the phbAB, fabH(F87W), and phaC1(SCQM) genes. The thermal and physical properties of the isolated polymers were characterized. It was found that even a very low mol % of MCL monomer in a SCL-MCL PHA copolymer had dramatic effects on the thermal properties of the copolymers.