Eutropha phb



Keywords: eutropha phb
Description: Polyhydroxyalkanoates (PHA) are biodegradable polymers that are attractive materials for use in tissue engineering and medical device manufacturing. Ralstonia eutropha is regarded as the model

Polyhydroxyalkanoates (PHA) are biodegradable polymers that are attractive materials for use in tissue engineering and medical device manufacturing. Ralstonia eutropha is regarded as the model organism for PHA biosynthesis. We examined the effects of PHA depolymerase (PhaZ) expression on PHA homeostasis in R. eutropha strains. In order to analyze the impact of PhaZs on R. eutropha granule architecture, we performed electron microscopy on several phaZ knockout strains and the wild type strain grown under PHA production conditions. Analysis of the acquired micrographs was based on stereology: the ratio of granule area and cell area was determined, along with total granule count per full-size cell image. Cells bearing a phaZ2 knockout mutation alone or in conjunction with a phaZ1 mutation were found to have a high granule volume per cell volume and a higher granule count compared to wild type. A phaZ quadruple knockout strain appeared to have a low granule volume per cell volume and a low granule count per cell. Cells bearing a phaZ3 knockout were found to have a higher granule count than the wild type, whereas granule volume per cell volume was similar. Accordingly, we hypothesize that PhaZs have not only an impact on PHA degradation but also on the 3-dimensional granule architecture. Based on our data, PhaZ2 is postulated to affect granule density. This work increased our knowledge about PHA depolymerases in R. eutropha. including enzymes that had previously been uncharacterized.

Ralstonia eutropha Polyhydroxyalkanoates Polyhydroxybutyrate Biomaterials Depolymerase Granules Carbon utilization Electron microscopy Stereology

The online version of this article (doi: 10.​1186/​2191-0855-2-26 ) contains supplementary material, which is available to authorized users.

Polyhydroxyalkanoates (PHA) are carbon storage molecules synthesized by many microorganisms during times of (non-carbon) nutrient stress. One of the most well-studied members of the PHA family is the homopolymer polyhydroxybutyrate (PHB). Several species of bacteria have been shown to produce PHB (see ([ Anderson and Dawes 1990 ]; [ Rehm 2003 ]; [ Stubbe et al. 2005 ]; [ Sudesh et al. 2000 ]) for reviews), including Ralstonia eutropha, which is considered the model organism for study of PHA biosynthesis ([ Reinecke and Steinbüchel 2009 ]). R. eutropha grows autotrophically ([ Ishizaki et al. 2001 ]; [ Volova and Voinov 2003 ]) or heterotrophically on carbon substrates such as fructose ([ Tian et al. 2005b ]), organic acids ([ Yang et al. 2010 ]), or fatty acids/lipids ([ Brigham et al. 2010 ]) and has been demonstrated to produce PHB under nitrogen or phosphate limitation. PHA, including but not limited to PHB, have been shown to exhibit a range of thermal and physical properties similar to petroleum-based plastics ([ Budde et al. 2011 ]; [ Sudesh et al. 2000 ]), and have also been shown to be biodegradable ([ Shishatskaya et al. 2005 ]).

Both the ubiquity of PHB, and various PHA types, and the production of low molecular weight PHB in a range of organisms, including mammals ([ Reusch 1989 ]), have led researchers to extensive examination of PHAs for use in medical materials and devices. PHAs have been demonstrated to be biocompatible and have been used in surgical procedures ([ Shishatskaya et al. 2002a ]; [ Shishatskaya et al. 2002b ]; [ Shishatskaya et al. 2004 ]; [ Sodian et al. 1999 ]). The immunological and histological responses of tissues and organisms towards PHA implants have demonstrated favorable results ([ Sevastianov et al. 2003 ]; [ Shishatskaya et al. 2004 ]; [ Volova et al. 2003 ]). PHA is currently being used to produce commercially available sutures and meshes that have shown to exhibit many promising characteristics, including durability, tensile strength, flexibility, and resorbability ( http://​www.​tepha.​com ). PHA matrices are also being examined for encapsulation of bioactive agents, such as tumor drugs and antibiotics, to be inserted and delivered at the site of tissue distress ([ Chee et al. 2008 ]). Many PHA types have been used as growth substrata for cells in tissue engineering ([ Chen and Wu 2005 ]). In earlier studies, a copolymer of polyglycolic acid (PGA) and PHB was used to produce pulmonary valve leaflets and pulmonary artery scaffolds in sheep ([ Shum-Tim et al. 1999 ]). This study was followed up by construction of a PHA-based heart valve scaffold, which was again surgically inserted into sheep ([ Sodian et al. 1999 ]). Both of these early studies illustrated that tissue engineering using biopolymer scaffolds is possible. Fibroblast cells from the NIH 3T3 line have been shown to exhibit growth on PHB and PHA copolymer substrates. These polymers have good functional properties in both an in vitro and in vivo setting ([ Shishatskaya et al. 2008 ]). PHB composites have been constructed, such as PHB with hydroxyapatite for bone tissue engineering ([ Chen and Wu 2005 ]). PHA, specifically a copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate (P(3HB-co-4HB)), shown to be a promising substrate for growing rabbit smooth muscle cells and demonstrated high elastin formation, suggesting efficacy of PHA in tissue engineering ([ Cheng et al. 2008 ]).

Even with the number of important discoveries that have been made regarding R. eutropha PHB metabolism, physiology, and biochemistry over the past few decades, there are still attributes of PHB metabolism and intracellular granule formation that remain to be fully explained. In this work, we examine PHB depolymerase enzymes, termed PhaZ, and their role in R. eutropha PHB utilization and intracellular PHB granule formation. Table 1 lists established and putative PHA depolymerases in R. eutropha. The principle depolymerase enzyme of R. eutropha. PhaZ1, has been well-studied and plays a significant role in PHB mobilization ([ Uchino and Saito 2006 ]; [ Uchino et al. 2007 ]; [ York et al. 2003 ]). Another depolymerase enzyme, PhaZ2, has been shown to degrade PHB intracellularly ([ York et al. 2003 ]). It is important to note that, in published research prior to publication of the R. eutropha H16 genome sequence in 2006, what is now catalogued as PhaZ5 was referred to as PhaZ3. A new and currently uncharacterized PhaZ3 enzyme is now referenced. Two depolymerase candidates, PhaZ3 and PhaZ5, exhibit homology to other intracellular depolymerases and are even classified in the PHA Depolymerase Engineering Database ( http://​www.​ded.​uni-stuttgart.​de/​ ) as intracellular, short-chain PHA depolymerases that act on native PHA ([ Knoll et al. 2009 ]). One candidate, PhaZ5 (formerly PhaZ3) has been discussed in previous works ( [ York et al.2003 ]), while PhaZ3 has not been characterized to date. Here, we describe the PHB mobilization phenotype of a phaZ3 mutant strain, alone or in conjunction with other phaZ gene deletions. We also examine expression of phaZ genes and determine if absence or overexpression of specific phaZ genes influences PHB homeostasis. Lastly, we examine phaZ mutant strains by electron microscopy and suggest PHB remodeling for PhaZ enzymes associated with PHB granules.






Photogallery Eutropha phb:


Examination of PHB Depolymerases in Ralstonia eutropha: Further ...


Design Considerations in Hybrid Neural Optimization of Fed-Batch ...


Biosynthesis of polyhydroxybutyrate (PHB) and extracellular ...


Mutation on N-terminus of polyhydroxybutyrate synthase of ...


Generation and Utilization of Microbial Biomass Hydrolysates in ...


Intelligent" descriptions of microbial kinetics in finitely ...


Poly(3-hydroxybutyrate) synthesis in fed-batch culture of ...


Comparison of phbC genes cloned from Ralstonia eutropha and ...


Production of polyhydroxybutyrate by Ralstonia eutropha from ...


Analysis of PHB Metabolism Applying Tn5 Mutagenesis in Ralstonia ...


The production of polyhydroxybutyrate by Methylobacterium ...


Production of polyhydroxyalkanoates from Ralstonia eutropha using ...


Lipid and fatty acid metabolism in Ralstonia eutropha: relevance ...


Repeated Batch Cultivation of Ralstonia eutropha for Poly ( ...


Kinetic analysis on formation of poly(3-hydroxybutyrate) from ...


Response coefficient analysis of a fed-batch bioreactor to ...


Site-directed saturation mutagenesis at residue F420 and ...


Characteristics of poly(3-hydroxybutyrate-co-4-hydroxybutyrate ...


Impact of each individual component of the mutated PTSNag on ...


Dynamics of Activity of the Key Enzymes of Polyhydroxyalkanoate ...


In vivo blending of medium chain length polyhydroxy-alkanoates and ...