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

Data de publicació electrònica: 11-11-2022Gallic 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

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) with a broad range of 3HV content at high yields by Burkholderia sacchari IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y-3HV/prop) increasing from 1.10 to 1.34 g g(-1). Copolymer productivity of 1 g l(-1) h(-1) was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y-3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg)

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

Abstract 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

Increasing PHB production with an industrially scalable hardwood hydrolysate as a carbon source

In this study, we report an industrially feasible conversion of wood hydrolysate from a pilot-scale forest biorefinery. The wood hydrolysate was compared to a synthetic hydrolysate with a similar content of carbon sources (glucose, xylose, and acetate). To maximize the PHB concentration, high-cell density cultivations were conducted in bioreactors for 52 h using the bacterium Paraburkholderia sacchari IPT 101 LMG 19450. The conversion of wood hydrolysate yielded a maximum PHB concentration of 34.5 g/L, which is among the highest reported for lignocellulose hydrolysates. In comparison, the use of a synthetic hydrolysate resulted in substantially lower PHB concentration (22.0 g/L). This could be attributed to a higher maximum specific growth rate of 0.36 vs. 0.33 per h for cells grown in wood hydrolysate vs. synthetic hydrolysate. For the wood hydrolysate, the final PHB content per cell mass reached 58 % g/g and a maximum PHB productivity of 0.72 g/(Lh), while the synthetic hydrolysate reached 55 % g/g and 0.46 g/(Lh) respectively. Given the increase in bacterial growth and PHB productivity when using the wood hydrolysate, the chosen lignocellulose conversion process is beneficial for subsequent biotechnological conversion to key bioproducts

Proteomic characterisation of toxins isolated from nematocysts of the South Atlantic jellyfish Olindias sambaquiensis

Surprisingly little is known of the toxic arsenal of cnidarian nematocysts compared to other\ud venomous animals. Here we investigate the toxins of nematocysts isolated from the jellyfish Olindias sambaquiensis. A total of 29 unique ms/ms events were annotated as potential toxins homologous to the toxic proteins from diverse animal phyla, including conesnails, snakes, spiders, scorpions, wasp, bee, parasitic worm and other Cnidaria. Biological activities of these potential toxins include cytolysins, neurotoxins, phospholipases and toxic peptidases. The presence of several toxic enzymes is intriguing, such as sphingomyelin phosphodiesterase B (SMase B) that has only been described in certain spider venoms, and a prepro-haystatin P-IIId snake venom metalloproteinase (SVMP) that activates coagulation factor X, which is very rare even in snake venoms. Our annotation reveals sequence orthologs to many representatives of the most important superfamilies of peptide venoms suggesting that their origins in higher organisms arise from deep eumetazoan innovations. Accordingly, cnidarian venoms may possess unique biological properties that might generate new leads in the discovery of novel pharmacologically active drugs.King’s College LondonSISBIO license 15031-2FAPESP (grant 2010/50174-7)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq