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.

During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the ␤-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g av ‫؍‬ 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15 N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both ␣-and ␤-positions, or only at the ␤-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the ␣-, ␤-, and methylene bridge positions. Deuterium or 15 N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-Shydrogen atom at the ␤-position of the propionate side chain by the 5-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3, 3, and 4.Modified tetrapyrroles such as hemes and chlorophylls play important roles in a range of essential life processes from respiration to photosynthesis. Their underlying molecular architecture is reflected in a shared biosynthetic pathway that requires the coordinated activity of a large number of highly diverse enzymes (1-3). During porphyrin formation two structurally unrelated coproporphyrinogen III oxidases catalyze the oxidative decarboxylation of the propionate side chains on pyrrole rings A and B of the macrocycle to the corresponding vinyl groups (Scheme 1a) (4). The oxygen-dependent enzyme, HemF, found in eukaryotes and some bacteria, uses molecular oxygen as an electron acceptor during this process (5). For oxygen-independent coproporphyrinogen formation most bacteria carry the oxygen-independent enzyme HemN. In Escherichia coli, HemN is a monomeric protein that contains an oxygen-sensitive [4Fe-4S] cluster (6, 7). The enzyme belo...

New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)-early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A-ESAT-6, recombinant Ag85A-ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1-and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A-ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A-ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine. Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), infects a wide range of hosts, including domestic livestock and wildlife, and also causes TB in humans. Bovine TB poses a public health risk, particularly in regions where pasteurization of milk is not routine. This is of particular concern because more than 94% of the world's population lives in such regions, and M. bovis is the causative agent for up to 10% of TB cases in humans in these regions (14). Bovine TB also has a considerable economic impact on the agricultural industry. The human TB vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) is only partially effective in both cattle and humans (2, 12). Development of an effective vaccine protecting against bovine TB would provide a cost-effective TB control strategy as well as have applicability for control of human TB caused by Mycobacterium tuberculosis.A number of new TB vaccines are entering human clinical trials, including recombinant BCG, virus-vectored vaccines, and recombinant protein vaccines (20). One of the major constraints in developing effective recombinant protein vaccines is t...

Vaccine delivery systems based on display of antigens on bioengineered bacterial polyester inclusions can stimulate cellular immune responses. The food-grade Gram-positive bacterium Lactococcus lactis was engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which abundantly displayed the hepatitis C virus core (HCc) antigen. In mice, the immune response induced by this antigen delivery system was compared to that induced by vaccination with HCc antigen displayed on PHB beads produced in Escherichia coli, to PHB beads without antigen produced in L. lactis or E. coli, or directly to the recombinant HCc protein.Vaccination site lesions were minimal in all mice vaccinated with HCc PHB beads or recombinant protein, all mixed in the oil-in-water adjuvant Emulsigen, while vaccination with the recombinant protein in complete Freund's adjuvant produced a marked inflammatory reaction at the vaccination site. Vaccination with the PHB beads produced in L. lactis and displaying HCc antigen produced antigen-specific cellular immune responses with significant release of gamma interferon (IFN-␥) and interleukin-17A (IL-17A) from splenocyte cultures and no significant antigen-specific serum antibody, while the PHB beads displaying HCc but produced in E. coli released IFN-␥ and IL-17A as well as the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and IL-6 and low levels of IgG2c antibody. In contrast, recombinant HCc antigen in Emulsigen produced a diverse cytokine response and a strong IgG1 antibody response. Overall it was shown that L. lactis can be used to produce immunogenic PHB beads displaying viral antigens, making the beads suitable for vaccination against viral infections.The food-grade Gram-positive bacterium, Lactococcus lactis has been increasingly considered as a production host for recombinant therapeutic proteins (6, 9, 49). The recent advances toward the development of efficient gene expression systems in L. lactis and the established safety profile of L. lactis based on long-term use in dairy food processing has led to new potential applications in protein production, therapeutic drug delivery, and vaccine delivery (5,27,30,38).Recently, it was shown that L. lactis can be engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which display the Staphylococcus aureus protein A-derived IgG binding region, the Z domain, and that these can be isolated for in vitro use in purification of IgG (26). This was achieved by establishing the PHB biosynthesis pathway in L. lactis and by overproducing a Z domain-PHB synthase fusion protein which remained attached to the PHB inclusion surface. The PHB synthase represents the only essential enzyme required for PHB inclusion formation (39,40). This strategy utilized protein engineering of the PHB synthase from Ralstonia eutropha for the display of various protein-based functions, such as technical enzymes, binding domains, or a fluorescent protein, at the surfaces of PHB beads as had been previously established in recombinant Escherichi...

Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.