Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, and PHB depolymerase A, respectively. Four of the five genes (phaZ1 to phaZ4) have been cloned, and one of them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Müller, and H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA depolymerase gene (phaZ5) was identified by colony hybridization of recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. The nucleotide sequence of a 3,704-bp EcoRI fragment was determined and found to contain two large open reading frames (ORFs) which coded for a polypeptide with significant similarities to glycerol-3-phosphate dehydrogenases of various sources (313 amino acids; M(r), 32,193) and for the precursor of PHB depolymerase A (PhaZ5; 433 amino acids; M(r), 44,906). The PHV depolymerase gene (phaZ4) was subcloned, and the nucleotide sequence of a 3,109-bp BamHI fragment was determined. Two large ORFs (ORF3 and ORF4) that represent putative coding regions were identified. The deduced amino acid sequence of ORF3 (134 amino acids; M(r), 14,686) revealed significant similarities to the branched-chain amino acid aminotransferase (IlfE) of enterobacteria. ORF4 (1,712 bp) was identified as the precursor of a PHV depolymerase (567 amino acids; M(r), 59,947). Analysis of primary structures of the five PHA depolymerases of P. lemoignei and of the PHB depolymerases of Alcaligenes faecalis and Pseudomonas pickettii revealed homologies of 25 to 83% to each other and a domain structure: at their N termini, they have typical signal peptides of exoenzymes. The adjacent catalytic domains are characterized by several conserved amino acids that constitute putative catalytic triads which consist of the consensus sequence of serine-dependent hydrolases including the pentapeptide G-X-S-X-G, a conserved histidine and aspartate, and a conserved region resembling the oxyanion hole of lipases. C terminal of the catalytic domain an approximately 40-amino-acid-long threonine-rich region (22 to 27 threonine residues) is present in PhaZ1, PhaZ2, PhaZ3, and PhaZ5. Instead of the threonine-rich region PhaZ4 and the PHB depolymerases of A. faecalis and P. pickettii contain an approximately 90-amino-acid-long sequence resembling the fibronectin type III module of eucaryotic extracellular matrix proteins. The function of the fibronectin type III module in PHA depolymerases remains obscure. Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5 and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polyactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca2+ and was inhibited by the presence of EDTA.
Polyhydroxybutyrates (PHBs) are polyoxoesters generated from (R)3-hydroxybutyryl coenzyme A by PHB synthase. During the polymerization reaction, the polymers undergo a phase transition and generate granules. Wautersia eutropha can transiently accumulate PHB when it is grown in a nutrient-rich medium (up to 23% of the cell dry weight in dextrose-free tryptic soy broth [TSB]). PHB homeostasis under these growth conditions was examined by quantitative Western analysis to monitor the proteins present, their levels, and changes in their levels over a 48-h growth period. The proteins examined include PhaC (the synthase), PhaP (a phasin), PhaR (a transcription factor), and PhaZ1a, PhaZ1b, and PhaZ1c (putative intracellular depolymerases), as well as PhaZ2 (a hydroxybutyrate oligomer hydrolase). The results show that PhaC and PhaZ1a were present simultaneously. No PhaZ1b or PhaZ1c was detected at any time throughout growth. PhaZ2 was observed and exhibited an expression pattern different from that of PhaZ1a. The levels of PhaP changed dramatically and corresponded kinetically to the levels of PHB. Transmission electron microscopy (TEM) provided the dimensions of the average cell and the average granule at 4 h and 24 h of growth (J. Tian, A. J. Sinskey, and J. Stubbe, J. Bacteriol. 187:3814-3824, 2005). This information allowed us to calculate the amount of each protein and number of granules per cell and the granule surface coverage by proteins. The molecular mass of PHB (106 Da) was determined by dynamic light scattering at 4 h, the time of maximum PHB accumulation. At this time, the surface area of the granules was maximally covered with PhaP (27 to 54%), and there were one or two PhaP molecules/PHB chain. The ratio of PHB chains to PhaC was ∼60, which required reinitiation of polymer formation on PhaC. The TEM studies of wild-type and ΔphaR strains in TSB provided further support for an alternative mechanism of granule formation (Tian et al., J. Bacteriol. 187:3814-3824, 2005).
Intracellular poly[d-(−)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share ∼30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (ΔphaZ1, ΔphaZ2, ΔphaZ3, ΔphaZ1ΔphaZ2, ΔphaZ1ΔphaZ3, ΔphaZ2ΔphaZ3, and ΔphaZ1ΔphaZ2ΔphaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated ∼80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.
Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser42, Asp138, and His178 were identified as catalytic-triad amino acids, with Ser42 as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.
Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHASCL) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596–607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHASCL depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [Mr], 43,610 Da) resembles precursors of other extracellular PHASCL depolymerases (28 to 50% identical amino acids). The mature protein (Mr, 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S136, D211, and H269 similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHASCL depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHASCL depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHASCL depolymerases.
An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2Reu) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2Reu. The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1Reu). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.
Degradation of poly(3-hydroxybutyrate) (PHB) by the thiolytic activity of the PHB depolymerase PhaZ1 from Ralstonia eutropha H16 was analyzed in the presence of different phasins. An Escherichia coli strain was constructed that harbored the genes for PHB synthesis (phaCAB), the phasin PhaP1, and the PHB depolymerase PhaZ1. PHB was isolated in the native form (nPHB) from this recombinant E. coli strain, and the in vitro degradation of the polyester was examined. Degradation resulted in the formation of the expected 3-hydroxybutyryl coenzyme A (3HB-CoA) and in the formation of a second product, which occurred in significantly higher concentrations than 3HB-CoA. This second product was identified by liquid chromatography mass spectrometry (LC-MS) as crotonyl-CoA. Replacement of PhaP1 by PhaP2 or PhaP4 resulted in a lower degradation rate, whereas the absence of the phasins prevented the degradation of nPHB by the PHB depolymerase PhaZ1 almost completely. In addition, the in vitro degradation of nPHB granules isolated from R. eutropha H16 (wild type) and from the R. eutropha ΔphaP1 and ΔphaP1-4 deletion mutants was examined. In contrast to the results obtained with nPHB granules isolated from E. coli, degradation of nPHB granules isolated from the wild type of R. eutropha yielded high concentrations of 3HB-CoA and low concentrations of crotonyl-CoA. The degradation of nPHB granules isolated from the ΔphaP1 and ΔphaP1-4 deletion mutants of R. eutropha was significantly reduced in comparison to that of nPHB granules isolated from wild-type R. eutropha. Stereochemical analyses of 3HB-CoA revealed that the (R) stereoisomer was collected after degradation of granules isolated from E. coli, whereas the (S) stereoisomer was collected after degradation of granules isolated from R. eutropha. Based on these results, a newly observed mechanism in the degradation pathway for PHB in R. eutropha is proposed which is connected by crotonyl-CoA to the β-oxidation cycle. According to this model, the NADPH-dependent synthesis of PHB with (R)-3HB-CoA as the intermediate and the PHB degradation yielding (S)-3HB-CoA, which is further converted in an NAD-dependent reaction, are separated.
Environmental microbes oscillate between feast and famine and need to carefully manage utilization, storage and conversion of reserve products to exploitable sources of carbon and energy. Polyhydroxyalkanoates (PHAs) are storage polymers that serve bacteria as sources of food materials under physiological conditions of carbon demand. In order to obtain insights into the role of PHA depolymerase (PhaZ) and its relationship to a PHA polymerase (PhaC2) in the carbon management activity of Pseudomonas putida strain U, we created a polymerase hyperexpression strain and a depolymerase knockout mutant of this strain, and examined their synthesis of PHA and expression of their PHA genes. This study revealed that hyperexpression of PhaC2 led to the accumulation of higher amounts of PHA (44%wt) than in the wild-type strain (24%wt) after 24 h of cultivation, which then returned to wild-type levels by 48 h, as a result of elevated depolymerization. The phaZ mutant, however, accumulated higher levels of PHA than the parental strain (62%wt), which were maintained for at least 96 h. Transcriptional analysis of the pha cluster by RT-PCR revealed that PHA operon proteins, including depolymerase, are expressed from the beginning of the growth phase. Hyperexpression of the PhaC2 polymerase was accompanied by an increase in the expression of the PhaZ depolymerase and a decrease in expression of another PHA polymerase, PhaC1. This suggests tight regulatory coupling of PHA polymerase and depolymerase activities that act in synergy, and in concert with other PHA proteins, to provide dynamic PHA granule synthesis and remodelling that rapidly and sensitively respond to changes in availability of carbon and the physiological-metabolic needs of the cell, to ensure optimal carbon resource management.
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.
Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary.
Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.
Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.
An intracellular poly[d(−)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric d(−)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.
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.
A gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome of Bacillus thuringiensis subsp. israelensis ATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designated phaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD of Pseudomonas putida was unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. The B. thuringiensis PhaZ was inactive against p-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-type B. thuringiensis cells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from a phaZ mutant lost this activity. The phaZ mutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate that B. thuringiensis harbors a new type of intracellular PHB depolymerase.
The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure.
Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.
Poly(3-hydroxybutyrate) (PHB) granules are organelle-like multienzyme-polymer complexes (carbonosomes) and are widespread storage compounds in prokaryotes. The interaction of three PHB granule-bound proteins (PHB synthase PhaC1, phasin PhaP5, and PHB/DNA binding protein PhaM) was studied in vivo by bimolecular fluorescence complementation (BiFC) microscopy in Ralstonia eutropha. To this end, a mobilizable 2-plasmid system for arabinose-controlled expression of protein fusions with the N-terminal (YN) and C-terminal (YC) parts of the enhanced yellow fluorescent protein (eYfp) in Gram-negative bacteria was developed. Both plasmids were stably expressed in Escherichia coli and in transconjugants of R. eutropha. Homo-oligomerization of PhaC1, PhaP5, and PhaM and interactions between PhaC1 and PhaM and between PhaM and PhaP5 were detected in R. eutropha and colocalized with PHB granules under PHB-permissive conditions. PhaM-PhaC1 complexes were detected near the midcell/nucleoid region in the absence of PHB. Expression of BiFC complexes in R. eutropha with PhaM (PhaM homo-oligomers or PhaM-PhaC1 or PhaM-PhaP5 complexes) resulted in substantial cell elongation compared to wild-type cells and in BiFC signals that were generally located near the midcell/nucleoid region. Western blot analysis of wild-type cell extracts and proteome analysis of PHB granule-bound proteins revealed that PhaM and PhaP5 are expressed in R. eutropha and that PhaM is constitutively expressed independently of the presence or absence of PHB. Size exclusion chromatography analysis in combination with cross-linking experiments of purified PhaP5-His6 and PhaM-His6 showed that PhaP5 forms dimers and that PhaM is present in oligomeric (dodecamer) form. Implications of this finding for subcellular PHB localization and initiation of PHB granule formation in R. eutropha will be discussed.
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.
Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.
A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.
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.
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.
The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Pp, class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA− as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB−4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB−4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB−4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.
A novel thermoalkalophilic depolymerase, PhaZ7, from P. lemoignei was crystallized by the microdialysis technique. Crystals belong to space group C2 and diffract to 2.75 Å resolution at a synchrotron source.
Polyhydroxyalkanoates (PHA) are biodegradable polyesters that have attracted commercial and academic interest as environmentally friendly materials. A number of enzymes are able to degrade polyhydroxyalkanoates to water-soluble products. PhaZ7 poly(3-hydroxybutyrate) (PHB) depolymerase (EC 22.214.171.124), a 342-amino-acid hydrolase from the PHA-degrading bacterium Paucimonas lemoignei, has been found to possess substrate specificity for amorphous PHA. PhaZ7 was crystallized by the microdialysis method. Thin rod-like crystals were grown in low ionic strength solution and found to belong to the monoclinic space group C2, with unit-cell parameters a = 225.8, b = 46.5, c = 171.3, β = 128.9°. A complete data set was collected to 2.75 Å resolution at 100 K using synchrotron radiation.
biopolymers; biodegradation; Paucimonas lemoignei; serine hydrolases; depolymerase
A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.
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 PhaP1Reu 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 PhaP1Reu which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1Reu or various regions of PhaP1Reu 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 PhaP1Reu 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 PhaP1Reu, which revealed that PhaP1Reu 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.