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.

Carriers based on poly-3-hydroxyalkanoates containing nanomagnetite to trigger hormone release.

Poly-3-hydroxybutyrate (P(3HB)) and poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(3HB-co-3HHx)) are biocompatible and bioabsorbable biopolymers produced by different bacteria with potential for drug delivery in thermo-responsive magnetic microcarriers. Microparticles of P(3HB) and P(3HB-co-3HHx), with 5.85% mol of 3HHx, produced by Burkholderia sacchari, containing nanomagnetite (nM) and lipophilic hormone were prepared by simple emulsion (oil/water) technique leading to progesterone (Pg) encapsulation efficiency higher than 70% and magnetite loads of 3.1 and 2.3% (w/w) for P(3HB)/nM/Pg and P(3HB-co-3HHx)/nM/Pg, respectively. These formulations were characterized by Infrared spectroscopy, X-ray diffraction, Thermal gravimetric analysis and Electron microscopy (TEM, SEM) techniques. The P(3HB)/nM/Pg and P(3HB-co-3HHx)/nM/Pg microparticles presented spherical geometry with wrinkled surfaces and average size between 2 and 40 µm for 90% of the microparticles. The release profiles of the P(3HB)/nM/Pg and P(3HB-co-3HHx)/nM/Pg formulations showed a hormone release trigger (6.9 and 11.1%, respectively) effect induced by oscillating external magnetic field (0.2 T), after 72 h. Progesterone release in non-magnetic tests with P(3HB-co-3HHx)/nM/Pg revealed a slight increment (5.6%) in relation to P(3HB)/nM/Pg. The experimental release of the P(3HB)/nM/Pg and P(3HB-co-3HHx)/nM/Pg samples presented a good agreement with Higuchi model. The 3HHx comonomer content improves the hormone release of the P(3HB-co-3HHx)/nM/Pg formulation with potential for application to synchronize the estrous cycle.

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.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from biodiesel by-product and propionic acid by mutant strains of Pandoraea sp.

Pandoraea sp. MA03 wild type strain was subjected to UV mutation to obtain mutants unable to grow on propionic acid (PA) but still able to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from glycerol and PA at high 3HV yields. In shake flask experiments, mutant prp25 was selected from 52 mutants affected in the propionate metabolism exhibiting a conversion rate of PA into 3HV units of 0.78 g g-1 . The use of crude glycerol (CG) plus PA or valeric acid resulted in a copolymer with 3HV contents varying from 21.9 to 30 mol% and 22.2 to 36.7 mol%, respectively. Fed-batch fermentations were performed using CG and PA and reached a 3HV yield of 1.16 g g-1 , which is 86% of the maximum theoretical yield. Nitrogen limitation was a key parameter for polymer accumulation reaching up to 63.7% content and 18.1 mol% of 3HV. Henceforth, mutant prp25 is revealed as an additional alternative to minimize costs and support the P(3HB-co-3HV) production from biodiesel by-products. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1077-1084, 2017.