Reviews Bacterial Polyhydroxyalkanoate Granules: Biogenesis, Structure, and Potential Use as Nano-/Micro-Beads in Biotechnological and Biomedical Applications Katrin Grage, † Anika C. Jahns, † Natalie Parlane, †,‡ Rajasekaran Palanisamy, † Indira A. Rasiah, † Jane A. Atwood, † and Bernd H. A. Rehm* ,† Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand and Hopkirk Research Institute, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand Received December 1, 2008; Revised Manuscript Received January 20, 2009 Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for industrial and biomedical applications. These polymers are synthesized by most bacteria in times of unbalanced nutrient availability from a variety of substrates and they are deposited intracellularly as insoluble spherical inclusions or PHA granules. The granules consist of a polyester core, surrounded by a boundary layer with embedded or attached proteins that include the PHA synthase, phasins, depolymerizing enzymes, and regulatory proteins. Apart from ongoing industrial interest in the material PHA, more recently there has also been increasing interest in applications of the PHA granules as nano-/micro-beads after it was conceived that fusions to the granule associated proteins (GAPs) provide a way to immobilize target proteins at the granule surface. This review gives an overview of PHA granules in general, including biogenesis and GAPs, and focuses on their potential use as nano-/micro-beads in biotechnological and biomedical applications. Introduction Bacterial polyhydroxyalkanoate (PHA) granules, which are found as naturally occurring spherical inclusions, are becoming increasingly recognized as potential functionalized beads for use in biotechnological and biomedical applications. PHAs are polyesters which serve as carbon and energy storage for bacteria and become deposited as insoluble spherical inclusions in the cytoplasm. Most bacterial genera and even members of the family Halobacteriaceae of the Archaea are known to synthesize PHA, 1-6 which is produced in conditions of nutrient limitation but where carbon is available in excess. 7-10 Bacteria are able to accumulate as much as 80% of their dry weight in PHA, 11,12 with reversal of the PHA polymerization process in conditions of carbon starvation. 13,14 One of the most common PHAs is poly(3-hydroxybutyrate) (PHB), which is synthesized from 3-hydroxybutyrate (3HB), but different bac- teria use hydroxy fatty acids of varying chain length, generating a range of PHAs. Due to properties such as biocompatibility, biodegradability, and production from renewable resources, there is considerable interest in the potential applications of PHAs. With chemical modification or through the creation of copolymers, a range of material properties can be achieved, for example, PHAs that are less brittle and more flexible while retaining tensile strength. These polymers have been developed for use in industrial or medical applications and have been shown to be well tolerated by mammalian systems. 15 Due to the comparatively high production costs, PHAs are currently mainly attractive for use in the medical field, for example, for sutures or implants like heart valves, stents, and bone scaffolding. 15,16 The key enzyme for PHA biosynthesis is the PHA synthase. This enzyme polymerizes (R)-3-hydroxyacyl-CoA thioester monomers into polyester with the release of coenzyme A. Depending on the organism, there are several classes of PHA synthases using different (R)-3-hydroxyacyl-CoA precursors that can be provided by different pathways. 17 In CupriaVidus necator, the most investigated PHB producer, 18 (R)-3-hydroxy- butyryl-CoA monomers are generated from acetyl-CoA by the action of two other enzymes. 16,19,20 The three PHB biosynthesis genes are organized in one operon, the phaCAB operon. -Ketothiolase (encoded by phaA) condenses two molecules of acetyl-CoA to acetoacetyl-CoA and this is subsequently reduced to (R)-3-hydroxybutyryl-CoA by the NADPH-dependent ac- etoacetyl-CoA reductase (encoded by phaB). The PHB synthase (encoded by phaC in C. necator) then converts the thioester monomers into the polyoxoester PHB. The polymer aggregates to form a spherical inclusion or granule of usually 50-500 nm in diameter with the amorphous hydrophobic PHA polyester at the core and attached or embedded proteins at the surface, including the PHA synthase, PHA depolymerases, structural, and regulatory proteins. 21,22 In this review, we summarize the current literature on PHA granules, their biogenesis and structure, and on protein engineer- ing approaches of associated proteins aiming at the design of PHA granules as biobeads for use in various biomedical applications. * To whom correspondence should be addressed. Phone: +64 6 350 5515, ext. 7890. Fax: +64 6 350 5688. E-mail: b.rehm@massey.ac.nz. † Institute of Molecular Biosciences. ‡ Hopkirk Research Institute. Biomacromolecules 2009, 10, 660–669 660 10.1021/bm801394s CCC: $40.75  2009 American Chemical Society Published on Web 03/10/2009