Genome-Wide Metabolic Reconstruction of the Synthesis of Polyhydroxyalkanoates from Sugars and Fatty Acids by Burkholderia Sensu Lato Species.

Burkholderia sensu lato (s.l.) species have a versatile metabolism. The aims of this review are the genomic reconstruction of the metabolic pathways involved in the synthesis of polyhydroxyalkanoates (PHAs) by Burkholderia s.l. genera, and the characterization of the PHA synthases and the pha genes organization. The reports of the PHA synthesis from different substrates by Burkholderia s.l. strains were reviewed. Genome-guided metabolic reconstruction involving the conversion of sugars and fatty acids into PHAs by 37 Burkholderia s.l. species was performed. Sugars are metabolized via the Entner-Doudoroff (ED), pentose-phosphate (PP), and lower Embden-Meyerhoff-Parnas (EMP) pathways, which produce reducing power through NAD(P)H synthesis and PHA precursors. Fatty acid substrates are metabolized via ß-oxidation and de novo synthesis of fatty acids into PHAs. The analysis of 194 Burkholderia s.l. genomes revealed that all strains have the phaC, phaA, and phaB genes for PHA synthesis, wherein the phaC gene is generally present in ≥2 copies. PHA synthases were classified into four phylogenetic groups belonging to class I II and III PHA synthases and one outlier group. The reconstruction of PHAs synthesis revealed a high level of gene redundancy probably reflecting complex regulatory layers that provide fine tuning according to diverse substrates and physiological conditions.

Identification of a self-sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2-hydroxyphenylacetic acid catabolism, via homogentisate pathway.

The self-sufficient cytochrome P450 RhF and its homologues belonging to the CYP116B subfamily have attracted considerable attention due to the potential for biotechnological applications based in their ability to catalyse an array of challenging oxidative reactions without requiring additional protein partners. In this work, we showed for the first time that a CYP116B self-sufficient cytochrome P450 encoded by the ohpA gene harboured by Cupriavidus pinatubonensis JMP134, a ß-proteobacterium model for biodegradative pathways, catalyses the conversion of 2-hydroxyphenylacetic acid (2-HPA) into homogentisate. Mutational analysis and HPLC metabolite detection in strain JMP134 showed that 2-HPA is degraded through the well-known homogentisate pathway requiring a 2-HPA 5-hydroxylase activity provided by OhpA, which was additionally supported by heterologous expression and enzyme assays. The ohpA gene belongs to an operon including also ohpT, coding for a substrate-binding subunit of a putative transporter, whose expression is driven by an inducible promoter responsive to 2-HPA in presence of a predicted OhpR transcriptional regulator. OhpA homologues can be found in several genera belonging to Actinobacteria and α-, ß- and γ-proteobacteria lineages indicating a widespread distribution of 2-HPA catabolism via homogentisate route. These results provide first time evidence for the natural function of members of the CYP116B self-sufficient oxygenases and represent a significant input to support novel kinetic and structural studies to develop cytochrome P450-based biocatalytic processes.

Influence of the carbon source on the properties of poly-(3)-hydroxybutyrate produced by Paraburkholderia xenovorans LB400 and its electrospun fibers.

Poly-3-hydroxybutyrate (PHB) is a biocompatible polymer produced by a wide variety of bacteria from different carbon sources. However, the carbon source effects on PHB properties are largely unknown. This study aimed to characterize PHB produced by Paraburkholderia xenovorans LB400 supplied with glucose (PHBg), mannitol (PHBm), or xylose (PHBx) as sole carbon sources and to evaluate their potential application as the main component of scaffolds obtained by electrospinning. The PHBs produced by strain LB400 had different molecular weights; the largest value corresponded to PHBm. The XRD-spectra revealed that PHB produced by strain LB400 from the three carbon sources are less crystalline than the commercially available polymer (PHBc). Moreover, the electrospinning process decreases even further their degree of crystallinity, which could lead to an improvement in the mechanical properties of the polymers. Relevantly, PHBx-microfibers exhibited mechanical characteristics similar to those of human skin. None of the scaffolds made of PHBs from strain LB400 grown in different carbon sources showed adverse effects on fibroblast cell growth. Thus, modifying the sugar used as the carbon source may be useful to tune the structural properties of PHB and its performance as a component of electrospun scaffolds, which may better fit specific biomedical applications.

Draft Genome Sequences of Two Pseudomonas Strains That Are Able To Use Furan Derivatives as Their Sole Carbon Source.

Pseudomonas sp. strains ALS1279 and ALS1131 were isolated from wastewater treatment facilities on the basis of their ability to use furfural, a key lignocellulose-derived inhibitor, as their only carbon source. Here, we present the draft genome sequences of both strains, which can shed light on catabolic pathways for furan compounds in pseudomonads.

Polyhydroxyalkanoates as biomaterial for electrospun scaffolds.

Polyhydroxyalkanoates (PHA) are natural polyesters produced by microorganisms under carbon source excess and limiting nutrient conditions. However, these biopolymers possess low mechanical and thermal properties, decreasing their potential applications in the medical field. Electrospinning is a technique that forms fibers from different polymers. PHA electrospun fibers improve the mechanical properties and decrease the crystallinity of PHA, including poly-3-hydroxybutyrate and its copolymers, which is attributed to the metastable structure (ß-form) formation. Therefore, the mechanical properties of fibers are intrinsically related to their plane orientation. Aligned fibers present better mechanical properties than randomly oriented fibers. However, randomly oriented fibers promote cell-fiber interaction and cell infiltration. Fibers produced with PHA blended with other polymers have shown improved mechanical and biological properties. Gelatin, zein and cellulose acetate are the main natural polymers that have been blended with PHA for electrospun scaffolds. For scaffold production by coaxial electrospinning, gelatin has been used as a shell and PHA as the core. PHA have been combined with different synthetic polymers and plasticizers resulting in an increase in the PHA miscibility. Therefore, the use of electrospinning in the development of PHA-based scaffolds seems to be an attractive method to change the intrinsic polymer features, increasing and enhancing PHA applications in tissue engineering.

Bacterial polyhydroxybutyrate for electrospun fiber production.

Nano- and microfibers obtained by electrospinning have attracted great attention due to its versatility and potential for applications in diverse technological fields. Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by microorganisms such as the bacterium Burkholderia xenovorans LB400. In particular, LB400 cells are capable to synthesize poly(3-hydroxybutyrate) (PHB) from glucose. The aim of this study was to produce and characterize electrospun fibers obtained from bacterial PHBs. Bacterial strain LB400 was grown in M9 minimal medium using xylose and mannitol (10gL-1) as the sole carbon sources and NH4Cl (1gL-1) as the sole nitrogen source. Biopolymer-based films obtained were used to produce fibers by electrospinning. Diameter and morphology of the microfibers were analyzed by scanning electron microscopy (SEM) and their thermogravimetric properties were investigated. Bead-free fibers using both PHBs were obtained with diameters of less than 3µm. The surface morphology of the microfibers based on PHBs obtained from both carbon sources was different, even though their thermogravimetric properties are similar. The results indicate that the carbon source may determine the fiber structure and properties. Further studies should be performed to analyze the physicochemical and mechanical properties of these PHB-based microfibers, which may open up novel applications.

Burkholderia xenovorans LB400 possesses a functional polyhydroxyalkanoate anabolic pathway encoded by the pha genes and synthesizes poly(3-hydroxybutyrate) under nitrogen-limiting conditions.

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that are synthesized by diverse bacteria. In this study, the synthesis of PHAs by the model aromatic-degrading strain Burkholderia xenovorans LB400 was analyzed. Twelve pha genes including three copies of phaC and five copies of the phasin-coding phaP genes are distributed among the three LB400 replicons. The phaC1ABR gene cluster that encodes the enzymes of the PHA anabolic pathway is located at chromosome 1 of strain LB400. During the growth of strain LB400 on glucose under nitrogen limitation, the expression of the phaC1, phaA, phaP1, phaR, and phaZ genes was induced. Under nitrogen limitation, PHA accumulation in LB400 cells was observed by fluorescence microscopy after Nile Red staining. GC-MS analyses revealed that the PHA accumulated under nitrogen limitation was poly(3-hydroxybutyrate) (PHB). LB400 cells grown on glucose as the sole carbon source under nitrogen limitation accumulated 40 ± 0.96% PHB of the cell dry weight, whereas no PHA was observed in cells grown in control medium. The functionality of the phaC1 gene from strain LB400 was further studied using heterologous expression in a Pseudomonas putida KT40C1ZC2 mutant strain derived from P. putida KT2440 that is unable to synthesize PHAs. Interestingly, KT40C1ZC2[pVNC1] cells that express the phaC1 gene from strain LB400 were able to synthesize PHB (33.5% dry weight). This study indicates that B. xenovorans LB400 possesses a functional PHA synthetic pathway that is encoded by the pha genes and is capable of synthesizing PHB.

Bacterial production of the biodegradable plastics polyhydroxyalkanoates.

Petroleum-based plastics constitute a major environmental problem due to their low biodegradability and accumulation in various environments. Therefore, searching for novel biodegradable plastics is of increasing interest. Microbial polyesters known as polyhydroxyalkanoates (PHAs) are biodegradable plastics. Life cycle assessment indicates that PHB is more beneficial than petroleum-based plastics. In this report, bacterial production of PHAs and their industrial applications are reviewed and the synthesis of PHAs in Burkholderia xenovorans LB400 is described. PHAs are synthesized by a large number of microorganisms during unbalanced nutritional conditions. These polymers are accumulated as carbon and energy reserve in discrete granules in the bacterial cytoplasm. 3-hydroxybutyrate and 3-hydroxyvalerate are two main PHA units among 150 monomers that have been reported. B. xenovorans LB400 is a model bacterium for the degradation of polychlorobiphenyls and a wide range of aromatic compounds. A bioinformatic analysis of LB400 genome indicated the presence of pha genes encoding enzymes of pathways for PHA synthesis. This study showed that B. xenovorans LB400 synthesize PHAs under nutrient limitation. Staining with Sudan Black B indicated the production of PHAs by B. xenovorans LB400 colonies. The PHAs produced were characterized by GC-MS. Diverse substrates for the production of PHAs in strain LB400 were analyzed.