Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media

Yarrowia lipolytica, a yeast strain with a huge biotechnological potential, capable to produce metabolites such as γ-decalactone, citric acid, intracellular lipids and enzymes, possesses the ability to change its morphology in response to environmental conditions. In the present study, a quantitative image analysis (QIA) procedure was developed for the identification and quantification of Y. lipolytica W29 and MTLY40-2P strains dimorphic growth, cultivated in batch cultures on hydrophilic (glucose and N-acetylglucosamine (GlcNAc) and hydrophobic (olive oil and castor oil) media. The morphological characterization of yeast cells by QIA techniques revealed that hydrophobic carbon sources, namely castor oil, should be preferred for both strains growth in the yeast single cell morphotype. On the other hand, hydrophilic sugars, namely glucose and GlcNAc caused a dimorphic transition growth towards the hyphae morphotype. Experiments for γ-decalactone production with MTLY40-2P strain in two distinct morphotypes (yeast single cells and hyphae cells) were also performed. The obtained results showed the adequacy of the proposed morphology monitoring tool in relation to each morphotype on the aroma production ability. The present work allowed establishing that QIA techniques can be a valuable tool for the identification of the best culture conditions for industrial processes implementation.

Production of secondary metabolites in cell suspension cultures of Ocimum sanctum L.

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepreneurship

Bioactivity of phenolic acids: Metabolites versus parent compounds: A review

Phenolic acids are present in our diet in different foods. In particular, mushrooms are a good source of these molecules. Due to their bioactive properties, phenolic acids are extensively studied and there is evidence of their role in disease prevention. Nevertheless, in vivo, these compounds are metabolized and circulate in the organism as glucuronated, sulfated and methylated metabolites, displaying higher or lower bioactivity. To clarify the importance of the metabolism of phenolic acids, the knowledge about the bioactivity of the metabolites is extremely important. In this review, chemical features, biosynthesis and bioavailability of phenolic acids are discussed as well as the chemical and enzymatic synthesis of their metabolites. Finally, the metabolites bioactive properties are compared with that of the corresponding parental compounds.

Lipids under stress - a lipidomic approach for the study of mood disorders

The emerging field of lipidomics has identified lipids as key players in disease physiology. Their physicochemical diversity allows precise control of cell structure and signaling events through modulation of membrane prop- erties and trafficking of proteins. As such, lipids are important regulators of brain function and have been implicated in neurodegenerative and mood disorders. Importantly, environmental chronic stress has been associated with anxiety and depression and its exposure in rodents has been extensively used as a model to study these diseases. With the accessibility to modern mass- spectrometry lipidomic platforms, it is now possible to snapshot the extensively interconnected lipid network. Here, we review the fundamentals of lipid biology and outline a framework for the interpretation of lipidomic studies as a new approach to study brain pathophysiology. Thus, lipid profiling provides an exciting avenue for the identification of disease signatures with important implications for diagnosis and treatment of mood disorders.

Synthetic biology approaches to engineer new pathways for the production of plant secondary metabolites

Secondary metabolites from plants are important sources of high-value chemicals, many of them being pharmacologically active. These metabolites are commonly isolated through inefficient extractions from natural biological sources and are often difficult to synthesize chemically. Therefore, their production using engineered organisms has lately attracted an increased attention. Curcuminoids, an example of such metabolites, are produced in Curcuma longa and exhibit anti-cancer and anti-inflammatory activities. Herein we report the construction of an artificial biosynthetic pathway for the curcuminoids production in Escherichia coli. Different 4-coumaroyl-CoA ligases (4CL) and polyketide synthases (diketide-CoA synthase (DCS), curcumin synthase (CURS) and curcuminoid synthase) were tested. The highest curcumin production (70 mg/L) was obtained by feeding ferulic acid and with the Arabidopsis thaliana 4CL1 and C. longa DCS and CURS enzymes. Other curcuminoids (bisdemethoxy- and demethoxycurcumin) were also produced by feeding coumaric acid or a mixture of coumaric and ferulic acids, respectively. Curcuminoids, including curcumin, were also produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase and 4-coumarate 3-hydroxylase were used. Caffeoyl-CoA O-methyltransferase was used to convert caffeoyl-CoA to feruloyl-CoA. This pathway represents an improvement of the curcuminoids heterologous production. The construction of this pathway in another model organism is being considered, as well as the introduction of alternative enzymes.

Lard as a suitable and cheap substrate for microbial lipids production by Yarrowia lipolytica

[Excerpt] Current agricultural and industrial practices have led to the generation of large amounts of various low-value or negative cost crude wastes, which are difficult and economically notattractive to treat and valorize. One important example of waste generation is animal fat, commonly found in tanning process and slaughterhouses. These wastes, in which the lipids are often the main and most problematic components, are not currently used effectively and there are almost no application methods to recover the respective value. (...)

Biotechnological valorization of waste cooking oils: lipase and microbial lipids production by Yarrowia lipolytica

[Excerpt] Waste cooking oils (WCO) generated from vegetable oils used at high temperatures in food frying, cause environmental problems and must be reutilized. New strategies to valorize these wastes are attracting a great scientific interest due to the important advantages offered from an economic and environmental point of view. A microbial platform can be established to convert low-value hydrophobic substrates, such as waste cooking oils, to microbial lipids (single cell oil, SCO) and other value-added bioproducts, such as lipase. (...)

Energy production from lipids by novel anaerobes

[Excerpt] Anaerobic microbial diversity encloses a very high potential that can be used for biotechnological applications. This potential is still largely unexplored, since the majority of the microorganisms in Nature are unknown or poorly characterized. This work is focused on the study of novel anaerobic microorganisms that participate in the metabolism of lipids, long chain fatty acids (LCFA) and glycerol, with the main goal of producing valuable energy-rich organic compounds. For that, conventional anaerobic culturing procedures were combined with continuous bioreactors operation and allied to microbial ecology approaches. Two main examples of the work performed will be presented. (...)