Orange and Passion Fruit Wastes Characterization, Substrate Hydrolysis and Cell Growth of Cupriavidus necator, as Proposal to Converting of Residues in High Value Added Product.

Brazil is the world's largest producer of orange and passion fruit, which are destined mainly for industrialization, generating grand volumes of wastes. The solid portion of these residues is a rich source of pectin - composed mainly of galacturonic acid and neutral sugars, which through the hydrolysis process can be used in biological conversion processes, as the production of polyhydroxyalkanoates (PHAs). This way, we characterized these wastes, followed by the extraction and hydrolysis of pectin for employ as a substrate for the cell growth of Cupriavidus necator. The results confirmed the large portion of pectin (almost 40 g.100g-1) and soluble sugars, present in these wastes. The hydrolyzed extract showed as a good source of carbon for the cell growth of C. necator with YX/S 0.56 and 0.44, µMax 0.27 and 0.21 for orange and passion fruit wastes respectively, similar to other carbon sources. This way, the extraction and hydrolysis of orange and passion fruit wastes for the cellular growth of C. necator, can be a good alternative to converting of residues in high value added product.

Expression and activity of the Calvin-Benson-Bassham cycle transcriptional regulator CbbR from Acidithiobacillus ferrooxidans in Ralstonia eutropha.

Autotrophic fixation of carbon dioxide into cellular carbon occurs via several pathways but quantitatively, the Calvin-Benson-Bassham cycle is the most important. CbbR regulates the expression of the cbb genes involved in CO2 fixation via the Calvin-Benson-Bassham cycle in a number of autotrophic bacteria. A gene potentially encoding CbbR (cbbR(AF)) has been predicted in the genome of the chemolithoautotrophic, extreme acidophile Acidithiobacillus ferrooxidans. However, this microorganism is recalcitrant to genetic manipulation impeding the experimental validation of bioinformatic predictions. Two novel functional assays were devised to advance our understanding of cbbR(AF) function using the mutated facultative autotroph Ralstonia eutropha H14 ΔcbbR as a surrogate host to test gene function: (i) cbbR(AF) was expressed in R. eutropha and was able to complement ΔcbbR; and (ii) CbbR(AF) was able to regulate the in vivo activity of four A. ferrooxidans cbb operon promoters in R. eutropha. These results open up the use of R. eutropha as a surrogate host to explore cbbR(AF) activity.

Influence of pH on the molecular weight of poly-3-hydroxybutyric acid (P3HB) produced by recombinant Escherichia coli.

The production of ultrahigh molecular weight poly-3-hydroxybutyric acid (P3HB) from carbohydrates by recombinant Escherichia coli harboring genes from Ralstonia eutropha was evaluated. In shaken-flask experiments, E. coli XL1 Blue harboring plasmid pSK::phaCAB produced P3HB corresponding to 40 and 27% of cell dry weight from glucose and xylose, respectively. Cultures in bioreactor using glucose as the sole carbon source at variable pH values (6.0, 6.5, or 7.0) allowed the production of P3HB with molecular weight varying between 2.0 and 2.5 MDa. These figures are significantly higher than the values often obtained by natural bacterial strains (0.5-1.0 MDa). Contrary to reports of other authors, no influence of pH was observed on the molecular weight of the polymer produced. Using xylose, P3HB with high molecular weight was also produced, indicating the possibility to produce these polymers from lignocellulosic materials.

Cupriavidus necator isolates are able to fix nitrogen in symbiosis with different legume species.

The aim of the present study was to identify a collection of 35 Cupriavidus isolates at the species level and to examine their capacity to nodulate and fix N(2). These isolates were previously obtained from the root nodules of two promiscuous trap species, Phaseolus vulgaris and Leucaena leucocephala, inoculated with soil samples collected near Sesbania virgata plants growing in Minas Gerais (Brazil) pastures. Phenotypic and genotypic methods applied for this study were SDS-PAGE of whole-cell proteins, and 16S rRNA and gyrB gene sequencing. To confirm the ability to nodulate and fix N(2), the presence of the nodC and nifH genes was also determined, and an experiment was carried out with two representative isolates in order to authenticate them as legume nodule symbionts. All 35 isolates belonged to the betaproteobacterium Cupriavidus necator, they possessed the nodC and nifH genes, and two representative isolates were able to nodulate five different promiscuous legume species: Mimosa caesalpiniaefolia, L. leucocephala, Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata. This is the first study to demonstrate that C. necator can nodulate legume species.

Involvement of several transcriptional regulators in the differential expression of tfd genes in Cupriavidus necator JMP134.

Cupriavidus necator JMP134 has been extensively studied because of its ability to degrade chloroaromatic compounds, including the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid (3-CB), which is achieved through the pJP4-encoded chlorocatechol degradation gene clusters: tfdCIDIEIFI and tfdDIICIIEIIFII. The present work describes a different tfd-genes expression profile depending on whether C. necator cells were induced with 2,4-D or 3-CB. By contrast, in vitro binding assays of the purified transcriptional activator TfdR showed similar binding to both tfd intergenic regions; these results were confirmed by in vivo studies of the expression of transcriptional lacZ fusions for these intergenic regions. Experiments aimed at investigating whether other pJP4 plasmid or chromosomal regulatory proteins could contribute to the differences in the response of both tfd promoters to induction by 2,4-D and 3-CB showed that the transcriptional regulators from the benzoate degradation pathway, CatR1 and CatR2, affected 3-CB- and 2,4-D-related growth capabilities. It was also determined that the ISJP4-interrupted protein TfdT decreased growth on 3-CB. In addition, an ORF with 34% amino acid identity to IclR-type transcriptional regulator members and located near the tfdII gene cluster module was shown to modulate the 2,4-D growth capability. Taken together, these results suggest that tfd transcriptional regulation in C. necator JMP134 is far more complex than previously thought and that it involves proteins from different transcriptional regulator families.