Patrícia Ferreira

Mentha piperita essential oil induces apoptosis in yeast associated with both cytosolic and mitochondrial ROS-mediated damage.

Mentha piperita (MP), also known as peppermint, is an aromatic and medicinal plant widely used in the food industry, perfumery and cosmetic, pharmacy and traditional medicine. Its essential oil (EO) displays antimicrobial activity against a range of bacteria and fungi. In this study, we found that MP EO lethal cytotoxicity is associated with increased levels of intracellular reactive oxygen species, mitochondrial fragmentation and chromatin condensation, without loss of the plasma membrane integrity, indicative of an apoptotic process. Overexpression of cytosolic catalase and superoxide dismutases reverted the lethal effects of the EO and of its major component menthol. Conversely, deficiency in Sod1p (cytosolic copper-zinc-superoxide dismutase) greatly increased sensitivity to both agents, but deficiency in Sod2p (mitochondrial manganese superoxide dismutase) only induced sensitivity under respiratory growth conditions. Mentha piperita EO increased the frequency of respiratory deficient mutants indicative of damage to the mitochondrial genome, although increase in mitochondrial thiol oxidation does not seem to be involved in the EO toxicity.

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project.

Oxygen transfer rate and pH as major operating parameters of citric acid production from glycerol by Yarrowia lipolytica W29 and CBS 2073

The amount of citric acid (CA) produced by Yarrowia lipolytica is dependent on the yeast strain and growth conditions such as pH, oxygen availability and medium composition. In this work, an experimental design based on the Taguchi method was applied to evaluate the effect of parameters: pH, carbon/nitrogen (C/N) ratio in the medium, oxygen mass transfer rate (OTR) and salts concentration, on the CA production by two Y. lipolytica strains, W29 (ATCC 20460) and CBS 2073. OTR and pH showed higher influence on the CA production for both strains. The increase of OTR from air to the culture medium led to a two- and three-fold improvement of the CA production by Y. lipolytica CBS 2073 and W29, respectively. Besides the individual effects of the parameters, a significant influence of the interaction between these parameters was observed, mainly between OTR and salts. Different values of the parameters were found at the optimum conditions for each strain, but the theoretically predicted and experimentally obtained citric acid concentrations (cCA) were approximately 10 g L−1 for both strains. The optimal conditions were also validated employing crude glycerol from biodiesel industry as a substrate, and similar behavior of the strains was observed.

Impact of the cultivation strategy on resveratrol production from glucose in engineered Corynebacterium glutamicum

The health benefits of polyphenols such as stilbenes and flavonoids for humans are increasingly attracting attention. Resveratrol is a well-characterized naturally-occurring stilbene and potent anti-oxidant, which is used as food supplement and cosmetic ingredient. Several microorganisms including Corynebacterium glutamicum were engineered for resveratrol production from glucose. Based on the cultivation of a resveratrol-producing C. glutamicum strain in shake flasks, different strategies for improving production under controlled conditions at bioreactor scale were tested. To this end, different cultivation parameters including substrate concentration and operation modes (batch and fed-batch) were evaluated. Whereas the highest biomass concentration was observed during fed-batch fermentation, the maximum resveratrol production was achieved in batch mode. The maximal titer obtained was 12mgL-1 of resveratrol without the addition of the fatty acid synthase inhibitor cerulenin, which was previously shown to be crucial for production with C. glutamicum. The specific growth rate during production seems to have a significant effect in resveratrol production and apparently low specific growth rates may redirect the metabolic bottleneck from p-coumaric acid formation to malonyl-CoA or ATP availability. We also show that high oxygen concentrations in the bioreactor negatively affected the obtained resveratrol titers with C. glutamicum, which is most likely due to the strong tendency of resveratrol to oxidize or oligomerize. Thus, up-scaling of the resveratrol production process is technically challenging and individual process parameters have to be optimized cautiously.

Heterologous production of resveratrol in bacterial hosts: current status and perspectives

The polyphenol resveratrol (3,5,4′-trihydroxystilbene) is a well-known plant secondary metabolite, commonly used as a medical ingredient and a nutritional supplement. Due to its health-promoting properties, the demand for resveratrol is expected to continue growing. This stilbene can be found in different plants, including grapes, berries (blackberries, blueberries and raspberries), peanuts and their derived food products, such as wine and juice. The commercially available resveratrol is usually extracted from plants, however this procedure has several drawbacks such as low concentration of the product of interest, seasonal variation, risk of plant diseases and product stability. Alternative production processes are being developed to enable the biotechnological production of resveratrol by genetically engineering several microbial hosts, such as Escherichia coli, Corynebacterium glutamicum, Lactococcus lactis, among others. However, these bacterial species are not able to naturally synthetize resveratrol and therefore genetic modifications have been performed. The application of emerging metabolic engineering offers new possibilities for strain and process optimization. This mini-review will discuss the recent progress on resveratrol biosynthesis in engineered bacteria, with a special focus on the metabolic engineering modifications, as well as the optimization of the production process. These strategies offer new tools to overcome the limitations and challenges for microbial production of resveratrol in industry.