Role of phaD in accumulation of medium-chain-length Poly(3-hydroxyalkanoates) in Pseudomonas oleovorans

Pseudomonas oleovorans is capable of producing poly(3-hydroxyalkanoates) (PHAs) as intracellular storage material. To analyze the possible involvement of phaD in medium-chain-length (MCL) PHA biosynthesis, we generated a phaD knockout mutant by homologous recombination. Upon disruption of the phaD gene, MCL PHA polymer accumulation was decreased. The PHA granule size was reduced, and the number of granules inside the cell was increased. Furthermore, mutant cells appeared to be smaller than wild-type cells. Investigation of MCL PHA granules revealed that the pattern of granule-associated proteins was changed and that the predominant protein PhaI was missing in the mutant. Complementation of the mutant with a phaD-harboring plasmid partially restored the wild-type characteristics of MCL PHA production and fully restored the granule and cell sizes. Furthermore, PhaI was attached to the granules of the complemented mutant. These results indicate that the phaD gene encodes a protein which plays an important role in MCL PHA biosynthesis. However, although its main effect seems to be the stabilization of MCL PHA granules, we found that the PhaD protein is not a major granule-associated protein and therefore might act by an unknown mechanism involving the PhaI protein.     

Role of phaD in Accumulation of Medium-Chain-Length Poly(3-Hydroxyalkanoates) in Pseudomonas oleovorans

Pseudomonas oleovorans is capable of producing poly(3-hydroxyalkanoates) (PHAs) as intracellular storage material. To analyze the possible involvement of phaD in medium-chain-length (MCL) PHA biosynthesis, we generated a phaD knockout mutant by homologous recombination. Upon disruption of the phaD gene, MCL PHA polymer accumulation was decreased. The PHA granule size was reduced, and the number of granules inside the cell was increased. Furthermore, mutant cells appeared to be smaller than wild-type cells. Investigation of MCL PHA granules revealed that the pattern of granule-associated proteins was changed and that the predominant protein PhaI was missing in the mutant. Complementation of the mutant with a phaD-harboring plasmid partially restored the wild-type characteristics of MCL PHA production and fully restored the granule and cell sizes. Furthermore, PhaI was attached to the granules of the complemented mutant. These results indicate that the phaD gene encodes a protein which plays an important role in MCL PHA biosynthesis. However, although its main effect seems to be the stabilization of MCL PHA granules, we found that the PhaD protein is not a major granule-associated protein and therefore might act by an unknown mechanism involving the PhaI protein.     

New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3‐hydroxybutyrate)

The metabolism of polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) has been investigated by many groups for about three decades, and good progress was obtained in understanding the mechanisms of biosynthesis and biodegradation of this class of storage molecules. However, the molecular events that happen at the onset of PHB synthesis and the details of the initiation of PHB/PHA granule formation, as well as the complex composition of the proteinaceous surface layer of PHB/PHA granules, have only recently come into the focus of research and were not reviewed yet. In this contribution, we summarize the progress in understanding the initiation and formation of the PHA granule complex at the example of Ralstonia eutropha H16 (model organism of PHB‐accumulating bacteria). Where appropriate, we include information on PHA granules of Pseudomonas putida as a representative species for medium‐chain‐length PHA‐accumulating bacteria. We suggest to replace the previous micelle mode of PHB granule formation by the Scaffold Model in which the PHB synthase initiation complex is bound to the bacterial nucleoid. In the second part, we highlight data on other forms of PHB: oligo‐PHB with ≈100 to 200 3‐hydroxybutyrate (3HB) units and covalently bound PHB (cPHB) are unrelated in function to storage PHB but are presumably present in all living organisms, and therefore must be of fundamental importance.     

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.     

聚羟基脂肪酸酯纳米微球：结构特征、生物合成及其在生物技术和生物医药领域的应用

聚羟基脂肪酸酯（polyhydroxyalkanoate）PHA 纳米微球是很多微生物在营养失衡的情况下,在体内合成的一种可生物降解的细胞内聚酯,主要作为微生物的碳源及能量储备。天然 PHA 微球的内部是由疏水的聚酯链构成的疏水核心,其外层是由磷脂界膜及膜上嵌入或附着的包括 PHA合酶 PhaC 和 PHA 颗粒相关蛋白 PhaP 等蛋白构成的边界层。PhaC 通过共价键连接在PHA微球表面,而 PhaP 通过疏水相互作用吸附在 PHA 微球表面。通过将外源性功能蛋白与 PhaC 或 PhaP 进行融合表达,在重组微生物体内就能直接合成表面带有功能蛋白的纳米微球复合体。由于该纳米微球在微生物细胞内是以独立的包涵体形式存在,因此通过细胞破碎及离心等方法就能简便、有效地使其从细胞中分离并得以纯化。鉴于 PHA 微球这种表面易被修饰改造的特性,越来越多的功能蛋白通过与 PHA 微球表面蛋白（PhaC 或 PhaP）的融合表达,呈递在了 PHA 微球表面,使其成为一种廉价、高效的蛋白固定化及呈递的新技术。本文在介绍了 PHA 微球的结构特性及生物合成的基础上,着重综述了目前关于功能化 PHA 微球在蛋白纯化、固定化酶、生物分离、靶向递药、疾病诊断、成像技术及新型疫苗开发方面的研究现状及其未来在生物医药等领域的广泛应用前景。     

Bacterial polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. More than 80 HAs have been detected as constituents of PHAs, which allows these thermoplastic materials to have various mechanical properties resembling hard crystalline polymer or elastic rubber depending on the incorporated monomer units. Even though PHAs have been recognized as good candidates for biodegradable plastics, their high price compared with conventional plastics has limited their use in a wide range of applications. A number of bacteria including Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinelandii, methylotrophs, pseudomonads, and recombinant Escherichia coli have been employed for the production of PHAs, and the productivity of greater than 2 g PHA/L/h has been achieved. Recent advances in understanding metabolism, molecular biology, and genetics of the PHA-synthesizing bacteria and cloning of more than 20 different PHA biosynthesis genes allowed construction of various recombinant strains that were able to synthesize polyesters having different monomer units and/or to accumulate much more polymers. Also, genetically engineered plants harboring the bacterial PHA biosynthesis genes are being developed for the economical production of PHAs. Improvements in fermentation/separation technology and the development of bacterial strains or plants that more efficiently synthesize PHAs will bring the costs down to make PHAs competitive with the conventional plastics. (c) 1996 John Wiley & Sons, Inc.     

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.     

The role of phasins in the morphogenesis of poly(3-hydroxybutyrate) granules

Recent developments in the understanding of the structure of polyhydroxyalkanoate, PHA, granules in bacteria are documented in the literature and point to the role of structural proteins, phasins, in granule formation and stabilization. We have previously conceived a computer program which successfully simulates granule formation in vitro, in the absence of phasins. Now we are extending the computer model to a more complex system, including phasins, to quantify their anticipated effect on the granule properties. The simulation enabled us to propose real experiments to test the validity of the model and provide a framework for a better understanding of PHA granule formation in vivo.     

Biosynthesis and mobilization of poly(3-hydroxybutyrate) [P(3HB)] by Spirulina platensis

Three strains of Spirulina platensis isolated from different locations showed capability of synthesizing poly(3-hydroxybutyrate) [P(3HB)] under nitrogen-starved conditions with a maximum accumulation of up to 10 wt.% of the cell dry weight (CDW) under mixotrophic culture conditions. Intracellular degradation (mobilization) of P(3HB) granules by S. platensis was initiated by the restoration of nitrogen source. This mobilization process was affected by both illumination and culture pH. The mobilization of P(3HB) was better under illumination (80% degradation) than in dark conditions (40% degradation) over a period of 4 days. Alkaline conditions (pH 10-11) were optimal for both biosynthesis and mobilization of P(3HB) at which 90% of the accumulated P(3HB) was mobilized. Transmission electron microscopy (TEM) revealed that the mobilization of P(3HB) involved changes in granule quantity and morphology. The P(3HB) granules became irregular in shape and the boundary region was less defined. In contrast to bacteria, in S. platensis the intracellular mobilization of P(3HB) seems to be faster than the biosynthesis process. This is because in cyanobacteria chlorosis delays the P(3HB) accumulation process.     

Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells

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.     

Advanced bacterial polyhydroxyalkanoates: Towards a versatile and sustainable platform for unnatural tailor-made polyesters

Polyhydroxyalkanoates (PHAs) are biopolyesters that generally consist of 3-, 4-, 5-, and 6-hydroxycarboxylic acids, which are accumulated as carbon and energy storage materials in many bacteria in limited growth conditions with excess carbon sources. Due to the diverse substrate specificities of PHA synthases, the key enzymes for PHA biosynthesis, PHAs with different material properties have been synthesized by incorporating different monomer components with differing compositions. Also, engineering PHA synthases using in vitro-directed evolution and site-directed mutagenesis facilitates the synthesis of PHA copolymers with novel material properties by broadening the spectrum of monomers available for PHA biosynthesis. Based on the understanding of metabolism of PHA biosynthesis, recombinant bacteria have been engineered to produce different types of PHAs by expressing heterologous PHA biosynthesis genes, and by creating and enhancing the metabolic pathways to efficiently generate precursors for PHA monomers. Recently, the PHA biosynthesis system has been expanded to produce unnatural biopolyesters containing 2-hydroxyacid monomers such as glycolate, lactate, and 2-hydroxybutyrate by employing natural and engineered PHA synthases. Using this system, polylactic acid (PLA), one of the major commercially-available bioplastics, can be synthesized from renewable resources by direct fermentation of recombinant bacteria. In this review, we discuss recent advances in the development of the PHA biosynthesis system as a platform for tailor-made polyesters with novel material properties.     

Influence of the poly-3-hydroxybutyrate (PHB) granule-associated proteins (PhaP1 and PhaP2) on PHB accumulation and symbiotic nitrogen fixation in Sinorhizobium meliloti Rm1021

Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.     

A Novel DNA-Binding Protein,PhaR, Plays a Central Role in the Regulation of Polyhydroxyalkanoate Accumulation and Granule Formation in the Haloarchaeon Haloferax mediterranei

Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei.     

Bioproduction of polyhydroxyalkanoates from bacteria: a metabolic approach

The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in Polyhydroxyalkanoates (PHAs) and the biosynthetic machinery for PHA metabolism has been the area of research over the last two decades. PHAs are polyesters of hydroxyalkanoates synthesized by numerous bacterial species with atleast five different PHA biosynthetic pathways. These are accumulated as an intracellular carbon and energy storage material. This diversity, in combination with genetic and molecular engineering has opened up this area for development of optimum PHA producing organisms. Even though PHAs have been recognized as a good candidate for biodegradable plastics, their industrial application is limited owing to high production cost. The classical microbiology and modern molecular biology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHA. This review provides an overview of the different PHA biosynthetic systems, the enzymes involved in PHA biosynthesis and there genetic background followed by a detailed summation of how this natural diversity is being used to develop commercially attractive recombinant process for large scale production of PHAs.     

Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic.

Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.     

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.     

A repressor protein,PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.