Exploring the potential of Burkholderia sacchari to produce polyhydroxyalkanoates.

AIM: Evaluation of the capability of Burkholderia sacchari to incorporate different monomers into polyhydroxyalkanoates (PHA). METHODS AND RESULTS: Thirty different carbon sources were evaluated as cosubstrates for B. sacchari growing on glucose with the intention to promote the incorporation of different monomers into the PHA produced by this species. With odd-numbered fatty acids, incorporation of the 3HV monomer was achieved, up to 65 mol% in the case of valerate. With 4-hydroxybutyrate, incorporation of 4HB was obtained, representing 9·1 mol%. With hexanoic acid, the production of P3HB-co-3HHx was achieved, containing up to 1·6 mol% of 3HHx. The molar fraction of 3HHx was found to be dependent on the ratio of glucose to hexanoic acid supplied. Metabolic flux analysis revealed a high efficiency of B. sacchari in converting carbon sources into P3HB-co-3HHx. Nevertheless, hexanoic acid was only poorly converted to 3HHx. CONCLUSIONS: Burkholderia sacchari is able to incorporate 3HV, 4HB and 3HHx in PHA containing mainly 3HB. The 3HHx content of P3HB-co-3HHx can be controlled by varying the glucose to hexanoic acid ratio. Burkholderia sacchari is highly efficient in converting carbon sources into PHA; however, only 2% of the hexanoic acid supplied could be converted to 3HHx. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing an approach to modulate the composition of P3HB-co-3HHx produced by bacteria using mixtures of carbohydrate and hexanoic acid as carbon source.

Role of CcpA in polyhydroxybutyrate biosynthesis in a newly isolated Bacillus sp. MA3.3.

The ccpA gene was inactivated in the polyhydroxybutyrate (PHB)-producing strain Bacillus sp. MA3.3 in order to reduce glucose catabolite repression over pentoses and develop improved bacterial strains for the production of PHB from lignocellulosic hydrolysates. Mutant Bacillus sp. MSL7 ΔCcpA are unable to grow on glucose and ammonia as sole carbon and nitrogen sources, respectively. Supplementation of glutamate as the nitrogen source or the substitution of the carbon source by xylose allowed the mutant to partially recover its growth performance. RT-PCR showed that CcpA stimulates the expression of the operon (gltAB),responsible for ammonia assimilation via glutamate in Bacillus sp. MA3.3. Moreover, it was demonstrated that the supplementation of xylose or glutamate was capable of stimulating gltAB operon expression independently of CcpA. In PHB production experiments in mineral media, it has been observed that the glucose catabolite repression over the pentoses was partially released in MSL7. Although the carbohydrate consumption is faster in the ccpA mutant, the biomass and PHB biosynthesis are lower, even with supplementation of glutamate. This is attributed to an increase of acetyl-CoA flux towards the tricarboxylic acid cycle observed in the mutant.

PHA(MCL) biosynthesis systems in Pseudomonas aeruginosa and Pseudomonas putida strains show differences on monomer specificities.

The production of PHA from plant oils by Pseudomonas species soil isolated from a sugarcane crop was evaluated. Out of 22 bacterial strains three were able to use efficiently plant oils to grow and to accumulate PHA. Pseudomonas putida and Pseudomonas aeruginosa strains produced PHA presenting differences on monomer composition compatible with variability on monomer specificity of their PHA biosynthesis system. The molar fraction of 3-hydroxydodecanoate detected in the PHA was linearly correlated to the oleic acid supplied. A non-linear relationship between the molar fractions of 3-hydroxy-6-dodecenoate (3HDdDelta(6)) detected in PHA and the linoleic acid supplied was observed, compatible with saturation in the biosynthesis system capability to channel intermediate of beta-oxidation to PHA synthesis. Although P. putida showed a higher 3HDdDelta(6) yield from linoleic acid when compared to P. aeruginosa, in both species it was less than 10% of the maximum theoretical value. These results contribute to the knowledge about the biosynthesis of PHA with a controlled composition from plant oils allowing in the future establishing the production of these polyesters as tailor-made polymers.

Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose, glucose and sugarcane bagasse hydrolysate.

Fifty-five bacterial strains isolated from soil were screened for efficient poly-3-hydroxybutyrate (P3HB) biosynthesis from xylose. Three strains were also evaluated for the utilization of bagasse hydrolysate after different detoxification steps. The results showed that activated charcoal treatment is pivotal to the production of a hydrolysate easy to assimilate. Burkholderia cepacia IPT 048 and B. sacchari IPT 101 were selected for bioreactor studies, in which higher polymer contents and yields from the carbon source were observed with bagasse hydrolysate, compared with the use of analytical grade carbon sources. Polymer contents and yields, respectively, reached 62% and 0.39 g g(-1) with strain IPT 101 and 53% and 0.29 g g(-1) with strain IPT 048. A higher polymer content and yield from the carbon source was observed under P limitation, compared with N limitation, for strain IPT 101. IPT 048 showed similar performances in the presence of either growth-limiting nutrient. In high-cell-density cultures using xylose plus glucose under P limitation, both strains reached about 60 g l(-1) dry biomass, containing 60% P3HB. Polymer productivity and yield from this carbon source reached 0.47 g l(-1) h(-1) and 0.22 g g(-1), respectively.

Identification of the 2-methylcitrate pathway involved in the catabolism of propionate in the polyhydroxyalkanoate-producing strain Burkholderia sacchari IPT101(T) and analysis of a mutant accumulating a copolyester with higher 3-hydroxyvalerate content.

Burkholderia sacchari IPT101(T) induced the formation of 2-methylcitrate synthase and 2-methylisocitrate lyase when it was cultivated in the presence of propionic acid. The prp locus of B. sacchari IPT101(T) is required for utilization of propionic acid as a sole carbon source and is relevant for incorporation of 3-hydroxyvalerate (3HV) into copolyesters, and it was cloned and sequenced. Five genes (prpR, prpB, prpC, acnM, and ORF5) exhibited identity to genes located in the prp loci of other gram-negative bacteria. prpC encodes a 2-methylcitrate synthase with a calculated molecular mass of 42,691 Da. prpB encodes a 2-methylisocitrate lyase. The levels of PrpC and PrpB activity were much lower in propionate-negative mutant IPT189 obtained from IPT101(T) and were heterologously expressed in Escherichia coli. The acnM gene (ORF4) and ORF5, which are required for conversion of 2-methylcitric acid to 2-methylisocitric acid in Ralstonia eutropha HF39, are also located in the prp locus. The translational product of ORF1 (prpR) had a calculated molecular mass of 70,598 Da and is a putative regulator of the prp cluster. Three additional open reading frames (ORF6, ORF7, and ORF8) whose functions are not known were located adjacent to ORF5 in the prp locus of B. sacchari, and these open reading frames have not been found in any other prp operon yet. In summary, the organization of the prp genes of B. sacchari is similar but not identical to the organization of these genes in other bacteria investigated recently. In addition, this study provided a rationale for the previously shown increased molar contents of 3HV in copolyesters accumulated by a B. sacchari mutant since it was revealed in this study that the mutant is defective in prpC.

Liofilizaçäo de leveduras de interesse industrial: influência das condiçöes de cultivo, velocidade de congelamento e meio protetor, na viabilidade celular após a liofilizaçäo / freeze-drying of industrial yeast strains: influence of growth conditions, coolong rates and suspending media on the viability of recovered cells

Dez linhagens de leveduras empregadas na produçäo de etanol no país foram estudadas, visando a obtençäo de maiores níveis de viabilidade celular após a liofilizaçäo.Foi testada a influência de três parâmetros:condiçöes de cultivo, velocidades de congelamento e meio protetor.Nos dois protetores testados, células cultivadas em meio sólido mostraram-se mais resistentes aos congelamentos rápido e lento do que aquelas crescidas sob agitaçäo.A velocidade de congelamento em si parece näo ser o único fator de morte celular.Engtretanto, os melhores resultados de viabilidade após a liofilizaçäo foram obtidos quando o congelamento lento foi previamente empregado.O emprego do congelamento rápido a células oriundos de culturas agitadas mostrou uma grande perda em viabilidade, evidenciada apenas após a liofilizaçäo.Assim sendo, embora esta forma de congelamento näo pareça causar perdas, o mesmo, quando associado a células cultivadas em shaker foi responsável pelos mais baixos valores de viabilidade celular.