From degrader to producer: reversing the gallic acid metabolism of Pseudomonas putida KT2440 / De degradador a productor: revirtiendo el metabolismo del ácido gálico de Pseudomonas putida KT2440

Gallic acid is a powerful antioxidant with multiple therapeutic applications, usually obtained from the acidic hydrolysis of tannins produced by many plants. As this process generates a considerable amount of toxic waste, the use of tannases or tannase-producing microorganisms has become a greener alternative over the last years. However, their high costs still impose some barriers for industrial scalability, requiring solutions that could be both greener and cost-effective. Since Pseudomonas putida KT2440 is a powerful degrader of gallic acid, its metabolism offers pathways that can be engineered to produce it from cheap and renewable carbon sources, such as the crude glycerol generated in biodiesel units. In this study, a synthetic operon with the heterologous genes aroG4, quiC and pobA* was developed and expressed in P. putida, based on an in silico analysis of possible metabolic routes, resulting in no production. Then, the sequences pcaHG and galTAPR were deleted from the genome of this strain to avoid the degradation of gallic acid and its main intermediate, the protocatechuic acid. This mutant was transformed with the vector containing the synthetic operon and was finally able to convert glycerol into gallic acid. Production assays in shaker showed a final concentration of 346.7 ± 0.004 mg L−1 gallic acid after 72 h.(AU)

From degrader to producer: reversing the gallic acid metabolism of Pseudomonas putida KT2440.

Gallic acid is a powerful antioxidant with multiple therapeutic applications, usually obtained from the acidic hydrolysis of tannins produced by many plants. As this process generates a considerable amount of toxic waste, the use of tannases or tannase-producing microorganisms has become a greener alternative over the last years. However, their high costs still impose some barriers for industrial scalability, requiring solutions that could be both greener and cost-effective. Since Pseudomonas putida KT2440 is a powerful degrader of gallic acid, its metabolism offers pathways that can be engineered to produce it from cheap and renewable carbon sources, such as the crude glycerol generated in biodiesel units. In this study, a synthetic operon with the heterologous genes aroG4, quiC and pobA* was developed and expressed in P. putida, based on an in silico analysis of possible metabolic routes, resulting in no production. Then, the sequences pcaHG and galTAPR were deleted from the genome of this strain to avoid the degradation of gallic acid and its main intermediate, the protocatechuic acid. This mutant was transformed with the vector containing the synthetic operon and was finally able to convert glycerol into gallic acid. Production assays in shaker showed a final concentration of 346.7 ± 0.004 mg L-1 gallic acid after 72 h.

Engineering Burkholderia sacchari to enhance poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] production from xylose and hexanoate.

Burkholderia sacchari LFM101 LMG19450T is a Brazilian bacterium isolated from sugarcane crops soil and a promising biotechnological platform for bioprocesses. It is an efficient producer of poly(3-hydroxybutyrate) from carbohydrates including xylose. In the present work, the expression of B. sacchari xylose consumption genes (xylA, xylB and tktA) was combined with the expression of Aeromonas sp. phaC (PHA synthase), aiming to increase both the growth rates in xylose and the 3-hydroxyhexanoate (3HHx) molar fractions in the produced PHA. Genes were cloned into pBBR1MCS-2 vectors and then expressed in the B. sacchari PHA- mutant LFM344. Maximum specific growth rates on xylose and PHA accumulation capacity of all recombinants were evaluated. In bioreactor experiments, up to 55.5 % CDW was accumulated as copolymer, hexanoate conversion to 3HHx raised from 2 % to 54 % of the maximum theoretical value, compared to wild type. 3HHx mol% ranged from 8 to 35, and molecular weights were between 111 and 220 kg/mol. Thermal analysis measurement showed a decrease in Tg and Tm values with higher 3HHx fraction, indicating improved thermomechanical characteristics. Recombinants construction and bioreactor strategies allowed the production of P(3HB-co-3HHx) with controlled monomeric composition from xylose and hexanoate, allowing its application in diverse fields, including the medical area.

Biopolymer production by halotolerant bacteria isolated from Caatinga biome.

Saline environments are extreme habitats with a high diversity of microorganisms source of a myriad of biomolecules. These microorganisms are assigned as extremophiles recognized to be producers of new natural compounds, which can be synthesized by helping to survive under harshness and extreme conditions. In Brazil, in the saline and semi-arid region of Areia Branca (Caatinga biome), halotolerant bacteria (able to growth at high NaCl concentrations) were isolated from rhizosphere of native plants Blutaparon portulacoides and Spergularia sp. and their biopolymer production was studied. A total of 25 bacterial isolates were identified at genus level based on 16S rRNA gene sequence analysis. Isolates were mainly Gram-positive bacteria from Bacillaceae, Staphylococcaceae, Microbacteriaceae, and Bacillales XII incertae sedis families, affiliates to Bacillus, Staphylococcus, Curtobacterium, and Exiguobacterium genera, respectively. One of the Gram-negative isolates was identified as member of the Pseudomonadaceae family, genus Pseudomonas. All the identified strains were halotolerant bacteria with optimum growth at 0.6-2.0 M salt concentrations. Assays for biopolymer production showed that the halotolerant strains are a rich source of compounds as polyhydroxyalkanoates (PHA), biodegradable biopolymer, such as poly(3-hydroxybutyrate) (PHB) produced from low-cost substrates, and exopolysaccharides (EPS), such as hyaluronic acid (HA), metabolite of great interest to the cosmetic and pharmaceutical industry. Also, eight bacterial EPS extracts showed immunostimulatory activity, promising results that can be used in biomedical applications. Overall, our findings demonstrate that these biomolecules can be produced in culture medium with 0.6-2.0 M NaCl concentrations, relevant feature to avoid costly production processes. This is the first report of biopolymer-producing bacteria from a saline region of Caatinga biome that showed important biological activities.

Exposure of Deinococcus radiodurans to both static magnetic fields and gamma radiation: observation of cell recuperation effects.

The extremophilic bacterium Deinococcus radiodurans displays an extraordinary ability to withstand lethal radiation effects, due to its complex mechanisms for both proteome radiation protection and DNA repair. Published results obtained recently at this laboratory show that D. radiodurans submitted to ionizing radiation results in its DNA being shattered into small fragments which, when exposed to a "static electric field' (SEF), greatly decreases cell viability. These findings motivated the performing of D. radiodurans exposed to gamma radiation, yet exposed to a different exogenous physical agent, "static magnetic fields" (SMF). Cells of D. radiodurans [strain D.r. GY 9613 (R1)] in the exponential phase were submitted to 60Co gamma radiation from a gamma cell. Samples were exposed to doses in the interval 0.5-12.5 kGy, while the control samples were kept next to the irradiation setup. Exposures to SMF were carried out with intensities of 0.08 T and 0.8 T delivered by two settings: (a) a device built up at this laboratory with niobium magnets, delivering 0.08 T, and (b) an electromagnet (Walker Scientific) generating static magnetic fields with intensities from 0.1 to 0.8 T. All samples were placed in a bacteriological incubator at 30 °C for 48 h, and after incubation, a counting of colony forming units was performed. Two sets of cell surviving data were measured, each in triplicate, obtained in independent experiments. A remarkable similarity between the two data sets is revealed, underscoring reproducibility within the 5% range. Appraisal of raw data shows that exposure of irradiated cells to SMF substantially increases their viability. Data interpretation strongly suggests that the increase of D. radiodurans cell viability is a sole magnetic physical effect, driven by a stochastic process, improving the efficiency of the rejoining of DNA fragments, thus increasing cell viability. A type of cut-off dose is identified at 10 kGy, above which the irradiated cellular system loses recovery and the cell survival mechanism collapses.

The relevance of enzyme specificity for coenzymes and the presence of 6-phosphogluconate dehydrogenase for polyhydroxyalkanoates production in the metabolism of Pseudomonas sp. LFM046.

Reconstruction of genome-based metabolic model is a useful approach for the assessment of metabolic pathways, genes and proteins involved in the environmental fitness capabilities or pathogenic potential as well as for biotechnological processes development. Pseudomonas sp. LFM046 was selected as a good polyhydroxyalkanoates (PHA) producer from carbohydrates and plant oils. Its complete genome sequence and metabolic model were obtained. Analysis revealed that the gnd gene, encoding 6-phosphogluconate dehydrogenase, is absent in Pseudomonas sp. LFM046 genome. In order to improve the knowledge about LFM046 metabolism, the coenzyme specificities of different enzymes was evaluated. Furthermore, the heterologous expression of gnd genes from Pseudomonas putida KT2440 (NAD+ dependent) and Escherichia coli MG1655 (NADP+ dependent) in LFM046 was carried out and provoke a delay on cell growth and a reduction in PHA yield, respectively. The results indicate that the adjustment in cyclic Entner-Doudoroff pathway may be an interesting strategy for it and other bacteria to simultaneously meet divergent cell needs during cultivation phases of growth and PHA production.

Investigating Nutrient Limitation Role on Improvement of Growth and Poly(3-Hydroxybutyrate) Accumulation by Burkholderia sacchari LMG 19450 From Xylose as the Sole Carbon Source.

Burkholderia sacchari LMG19450, a non-model organism and a promising microbial platform, was studied to determine nutrient limitation impact on poly(3-hydroxybutyrate) [P(3HB)] production and bacterial growth from xylose, a major hemicellulosic residue. Nitrogen and phosphorus limitations have been studied in a number of cases to enhance PHA accumulation, but not combining xylose and B. sacchari. Within this strategy, it was sought to understand how to control PHA production and even modulate monomer composition. Nitrogen-limited and phosphorus-limited fed-batch experiments in bioreactors were performed to evaluate each one's influence on cell growth and poly(3-hydroxybutyrate) production. The mineral medium composition was defined based on yields calculated from typical results so that nitrogen was available during phosphorus limitation and residual phosphorus was available when limiting nitrogen. Sets of experiments were performed so as to promote cell growth in the first stage (supplied with initial xylose 15 g/L), followed by an accumulation phase, where N or P was the limiting nutrient when xylose was fed in pulses to avoid concentrations lower than 5 g/L. N-limited fed-batch specific cell growth (around 0.19 1/h) and substrate consumption (around 0.24 1/h) rates were higher when compared to phosphorus-limited ones. Xylose to PHA yield was similar in both conditions [0.37 gP(3HB)/gxyl]. We also described pst gene cluster in B. sacchari, responsible for high-affinity phosphate uptake. Obtained phosphorus to biomass yields might evidence polyphosphate accumulation. Results were compared with studies with B. sacchari and other PHA-producing microorganisms. Since it is the first report of the mentioned kinetic parameters for LMG 19450 growing on xylose solely, our results open exciting perspectives to develop an efficient bioprocess strategy with increased P(3HB) production from xylose or xylose-rich substrates.

Engineering xylose metabolism for production of polyhydroxybutyrate in the non-model bacterium Burkholderia sacchari.

BACKGROUND: Despite its ability to grow and produce high-value molecules using renewable carbon sources, two main factors must be improved to use Burkholderia sacchari as a chassis for bioproduction at an industrial scale: first, the lack of molecular tools to engineer this organism and second, the inherently slow growth rate and poly-3-hydroxybutyrate [P(3HB)] production using xylose. In this work, we have addressed both factors. RESULTS: First, we adapted a set of BglBrick plasmids and showed tunable expression in B. sacchari. Finally, we assessed growth rate and P(3HB) production through overexpression of xylose transporters, catabolic or regulatory genes. Overexpression of xylR significantly improved growth rate (55.5% improvement), polymer yield (77.27% improvement), and resulted in 71% of cell dry weight as P(3HB). CONCLUSIONS: These values are unprecedented for P(3HB) accumulation using xylose as a sole carbon source and highlight the importance of precise expression control for improving utilization of hemicellulosic sugars in B. sacchari.

A non-naturally-occurring P(3HB-co-3HAMCL) is produced by recombinant Pseudomonas sp. from an unrelated carbon source.

Pseudomonas sp. PHA- was used as host for PHA biosynthesis genes from Aeromonas sp. to produce 3HB-co-3HAMCL from glucose with no supply of co-substrates. A non-naturally-occurring PHA composed mainly of 3HB, 3HHx and 3HD (3HO, 3HDdΔ5 and 3HDd monomers were detected in smaller amounts) was obtained. The polymer was extracted using two different solvents (acetone and chloroform) and subject to the following characterization tests: FTIR, DSC, TGA and GPC. The latter suggests a block copolymer since a single and narrow elution peak was observed for each sample. The DSC results ruled out the possibility of a random copolymer and agrees with a single copolymer composed of two blocks: one with the typical composition of PHAMCL produced by Pseudomonas and another containing 3HB and 3HHx with a high 3HHx molar fraction. Thus, this study increases the perspectives of P(3HB-co-3HAMCL) production from carbohydrates as the sole carbon source.

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures.

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHAMCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HAMCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HAMCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

Combining molecular and bioprocess techniques to produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with controlled monomer composition by Burkholderia sacchari.

Biopolymers as polyhydroxyalkanoates (PHA) composed by different co-monomers 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] has attracted interest since its properties are similar to low density polyethylene. Burkholderia sacchari produces this copolymer with a very low 3HHx molar fraction, about 2 mol%. B. sacchari mutant (unable to produce polymer) was engineered to host PHA biosynthesis genes (phaPCJ) from Aeromonas sp. In addition, a two-step bioprocess to increase biopolymer production was developed. The combination of these techniques resulted in the production of P(3HB-co-3HHx) with 3HHx content up to 20 mol%. The PHA content was about 78% of dry biomass, resulting in PHA volumetric productivities around 0.45gl-1h-1. The P(3HB-co-3HHx) containing 20 mol% of 3HHx presented an elongation at brake of 945%, higher than reported before for this PHA composition. Here we have described an approach to increase 3HHx content into the copolymer, allowing the precise control of the 3HHx molar fractions.

A suitable procedure to choose antimicrobials as controlling agents in fermentations performed by bacteria

This work evaluated the influence of nitrofurantoin, erythromycin and streptomycin at 50, 25 and 12,5 (per cent) of the minimal inhibitory concentration (MIC) on maximum specific growth rate (µmax) and specific polymer accumulation rate (µPHB) of "Alcaligenes eutrophus", considered resistant to those antimicrobials. Nitrofurantoin strongly affected µmax even at 50(per cent) MIC. Streptomycin moderately affected µmax only at 50(per cent) MIC. Nitrofurantoin showed the most harmful effect on µPHB when 50(per cent) MIC was applied and erythromycin was not harmful.