Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha.

Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)], a flexible and practical kind of polyhydroxyalkanoates, is generally produced from plant oils and fatty acids by several wild and recombinant bacteria. This study established an improved artificial pathway for the biosynthesis of P(3HB-co-3HHx) with high 3HHx composition from structurally unrelated fructose in Ralstonia eutropha. Depression of (R)-specific reduction of acetoacetyl-CoA by the deletion of phaB1 was an effective modification for formation of the C6-monomer unit from fructose driven by crotonyl-CoA carboxylase/reductase (Ccr). Co-overexpression of phaJ4a, which encodes medium-chain-length (R)-enoyl-CoA hydratase, with ccr promoted the incorporation of both 3HB and 3HHx units. Further introduction of emdMm, a synthetic gene encoding ethylmalonyl-CoA decarboxylase derived from mouse, was remarkably effective for P(3HB-co-3HHx) biosynthesis, probably by converting ethylmalonyl-CoA generated by the reductive carboxylase activity of Ccr back into butyryl-CoA. A high cellular content of P(3HB-co-3HHx) composed of 22mol% 3HHx could be produced from fructose by the engineered strain of R. eutropha with ΔphaB1 genotype expressing ccr, phaJ4a, and emd.

Modification of ß-oxidation pathway in Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from soybean oil.

Ralstonia eutropha H16 is a useful platform for metabolic engineering aiming at efficient production of polyhydroxyalkanaotes being attracted as practical bioplastics. This study focused on bifunctional (S)-specific 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase encoded by fadB to obtain information regarding ß-oxidation in this bacterium and to achieve compositional regulation of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] synthesized from soybean oil. In addition to two FadB homologs (FadB1 and FadB') encoded within the previously identified ß-oxidation gene clusters on the chromosome 1, a gene of third homolog (FadB2) was found on chromosome 2 of R. eutropha. The fadB homologs were disrupted in R. eutropha strain NSDG expressing a mutant gene of PHA synthase from Aeromonas caviae. The gene disruptions affected neither growth nor PHA production on fructose. On soybean oil, fadB' deletion led to reduction of PHA quantity attributed to decrease of 3HB unit, while fadB1 deletion slightly increased 3HHx composition without serious negative impact on both cell growth and PHA biosynthesis. Double deletion of fadB1 and fadB' significantly impaired the cell growth and PHA biosynthesis, indicating the major roles of fadB1 and fadB' in ß-oxidation. When fadB1 was deleted in several engineered strains of R. eutropha possessing additional (R)-enoyl-CoA hydratase gene(s), the net amounts of 3HHx unit in the PHA fractions showed 6-21% increase probably due to slightly enhanced supply of medium-chain-length 2-enoyl-CoAs through the partially impaired ß-oxidation. These results demonstrated that modification of ß-oxidation by fadB1 deletion was effective for increasing 3HHx composition in the copolyesters produced from soybean oil.

Characterization and functional analyses of R-specific enoyl coenzyme A hydratases in polyhydroxyalkanoate-producing Ralstonia eutropha.

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-fold-higher 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 ß-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.

Vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis.

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.

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16.

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-ß-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.

Production of functionalized biopolyester granules by recombinant Lactococcus lactis.

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 nisin-controlled 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 granule-associated 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.