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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...

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P(lac), P(tac), or P(BAD) derived from Escherichia coli, or promoter regions of phaC1 (P(phaC)) or phaP1 (P(phaP)) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P(lac), P(tac), P(phaC), and P(phaP ) mediated constitutive gene expression, among which P(tac) was the strongest promoter. lacI-P(tac) was not thoroughly functional even after addition of isopropyl-β-D-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P(BAD) could be regulated by varying L-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P(phaP) exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P(phaP ) for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.

A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R-specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded by phaJ4 from Pseudomonas aeruginosa (phaJ4 Pa ). The recombinant forms of the three proteins, termed PhaJ4a Re to PhaJ4c Re , actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C 4 to C 8 . PhaJ4a Re and PhaJ4b Re showed >10-foldhigher catalytic efficiency than PhaJ4c Re . The functions of the new PhaJ4 proteins were investigated using previously engineered R. eutropha strains as host strains; these strains are capable of synthesizing poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Deletion of phaJ4a Re from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4b Re or phaJ4c Re , indicating that only PhaJ4a Re was one of the major enzymes supplying the (R)-3HHx-CoA monomer through ␤-oxidation. Introduction of phaJ4a Re or phaJ4b Re into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4c Re did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co-3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially when phaJ4a Re or phaJ4b Re was tandemly introduced with phaJ Ac from Aeromonas caviae.

Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisincontrolled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granuleassociated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.

Previous studies have demonstrated that heterologous expression of PHA synthase from Aeromonas caviae (PhaCAc), capable of accepting (R)-3-hydroxyacyl-CoA of C4–C7 as substrates, could confer the ability to PHA-negative mutant of Cupriavidus necator PHB-4 to synthesize poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from vegetable oils. The mutation point within pha operon in PHB-4 was determined to be a single nonsense mutation within the PHA synthase gene (phaCCn), suggesting the much lower β-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase activities observed in this strain would be a polar effect of the mutation. For further efficient biosynthesis of P(3HB-co-3HHx) copolyester, C. necator wild strain H16 was engineered by homologous recombination targeting the chromosomal phaCCn, and the PHA productivity was compared with previous PHB–4-derived strain harboring phaCAc on a multi-copy plasmid (PHB–4/pJRDEE32d13). A strain H16CAc, in which phaCCn was substituted for phaCAc on the chromosome, could produce P(3HB-co-3HHx) from soybean oil with high productivity, but the 3HHx fraction in the accumulated polymer was decreased. Meanwhile, H16ΔC/pJRDEE32d13, that lost region for the original synthase gene and expresses exochromosomal phaCAc, grew and accumulated PHA with similar properties to the PHB–4-derived strain. The results of enzyme assay suggested that low β-ketothiolase activity might be relevant for decrease of growth ability accompanied by increase of 3HHx composition when soybean oil was fed as a sole carbon source. Key words: poly(hydroxyalkanoates), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHA synthase, Cupriavidus necator, vegetable oil.

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