The Model System Saccharomyces cerevisiae Versus Emerging Non-Model Yeasts for the Production of Biofuels.

Microorganisms are effective platforms for the production of a variety of chemicals including biofuels, commodity chemicals, polymers and other natural products. However, deep cellular understanding is required for improvement of current biofuel cell factories to truly transform the Bioeconomy. Modifications in microbial metabolic pathways and increased resistance to various types of stress caused by the production of these chemicals are crucial in the generation of robust and efficient production hosts. Recent advances in systems and synthetic biology provide new tools for metabolic engineering to design strategies and construct optimal biocatalysts for the sustainable production of desired chemicals, especially in the case of ethanol and fatty acid production. Yeast is an efficient producer of bioethanol and most of the available synthetic biology tools have been developed for the industrial yeast Saccharomyces cerevisiae. Non-conventional yeast systems have several advantageous characteristics that are not easily engineered such as ethanol tolerance, low pH tolerance, thermotolerance, inhibitor tolerance, genetic diversity and so forth. Currently, synthetic biology is still in its initial steps for studies in non-conventional yeasts such as Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris. Therefore, the development and application of advanced synthetic engineering tools must also focus on these underexploited, non-conventional yeast species. Herein, we review the basic synthetic biology tools that can be applied to the standard S. cerevisiae model strain, as well as those that have been developed for non-conventional yeasts. In addition, we will discuss the recent advances employed to develop non-conventional yeast strains that are efficient for the production of a variety of chemicals through the use of metabolic engineering and synthetic biology.

Methodologies and Applications of Proteomics for Study of Yeast Strains: An Update.

Yeasts are one of the mostly used microorganisms as models in several studies. A wide range of applications in different processes can be attributed to their intrinsic characteristics. They are eukaryotes and therefore valuable expression hosts that require elaborate post-translational modifications. Their arsenal of proteins has become a valuable biochemical tool for the catalysis of several reactions of great value to the food (beverages), pharmaceutical and energy industries. Currently, the main challenge in systemic yeast biology is the understanding of the expression, function and regulation of the protein pool encoded by such microorganisms. In this review, we will provide an overview of the proteomic methodologies used in the analysis of yeasts. This research focuses on the advantages and improvements in their most recent applications with an understanding of the functionality of the proteins of these microorganisms, as well as an update of the advances of methodologies employed in mass spectrometry.

Transcriptional profile of a bioethanol production contaminant Candida tropicalis.

The fermentation process is widely used in the industry for bioethanol production. Even though it is widely used, microbial contamination is unpredictable and difficult to control. The problem of reduced productivity is directly linked to competition for nutrients during contamination. Yeasts representing the Candida species are frequently isolated contaminants. Elucidating the behavior of a contaminant during the fermentation cycle is essential for combatting the contamination. Consequently, the aim of the current study was to better understand the functional and transcriptional behavior of a contaminating yeast Candida tropicalis. We used a global RNA sequencing approach (RNA-seq/MiSeq) to analyze gene expression. Genes with significantly repressed or induced expression, and related to the fermentations process, such as sugar transport, pyruvate decarboxylase, amino acid metabolism, membrane, tolerance to high concentrations of ethanol and temperatures, nutrient suppression), and transcription-linked processes, were identified. The expression pattern suggested that the functional and transcriptional behavior of the contaminating yeast during fermentation for bioethanol production is similar to that of the standard yeast Saccharomyces cerevisiae. In addition, the analysis confirmed that C. tropicalis is an important contaminant of the alcoholic fermentation process, generating bioethanol and viability through its tolerance to all the adversities of a fermentation process essential for the production of bioethanol. According on the gene expression profile, many of these mechanisms are similar to those of S. cerevisiae strains currently used for bioethanol production. These mechanisms can inform studies on antimicrobials, to combat yeast contamination during industrial bioethanol production.

Physiology of Lichtheimia ramosa obtained by solid-state bioprocess using fruit wastes as substrate.

Due to the amount of nutrients available in the agroindustrial wastes, these can be converted into high added-value products by the action of microorganisms in solid-state bioprocesses. The aim of this work was to evaluate the growth physiology and lipase production of the fungus Lichtheimia ramosa using the following Brazilian savannah fruit wastes as substrates: bocaiuva (Acrocomia aculeata), pequi (Caryocar brasiliense), guavira (Campomanesia pubescens), araticum (Annona crassiflora) and seriguela (Spondias purpurea). These residues were triturated, homogenized, adjusted to pH 5.0 and 60 % moisture, sterilized and packaged in plastic tray-type bioreactors before inoculation with 10 % (w/v) of L. ramosa pre-culture medium. The cultivations were conducted in a bacteriological incubator at 30 °C for 40 days. Samples were taken every 5 days and fungi and bacteria contents, proximate composition and lipase activity were evaluated. The maximum fungal counting was observed between 25 and 35 days. L. ramosa reached the stationary phase next to 40 days in all substrates. Mesophilic and psicrophilic aerobic bacteria were not detected. Protein enrichment was obtained for all media, being superior in seriguela residues (391.66 %), followed by pequi (160.04 %), araticum (143.31 %), guavira (102.42 %), and bocaiuva (67.88 %). Lipase production was observed in all cultivated media, except in pequi residues that showed decreasing lipase activity. The higher production was observed in guavira (1.12 U/g) followed by araticum (0.58 U/g), seriguela (0.41 U/g) and bocaiuva (0.21 U/g) waste substrates. It was concluded that the studied fruit wastes have been successfully utilized as substrates for protein enrichment and lipase production with L. ramosa.

Production of enzymes from Lichtheimia ramosa using Brazilian savannah fruit wastes as substrate on solid state bioprocessess

Background: Enzyme production by solid state bioprocess (SSB) using residues as substrate for microorganisms is an alternative for costs reduction and to avoid their disposal into environment. The aim of this work was to evaluate the physiology of the fungus Lichtheimia ramosa in terms of microbial growth and production of amylases, β-glucosidases, carboxymethylcellulase (CMCase), and xylanases, via SSB, utilizing wastes of the Brazilian savannah fruits bocaiuva (Acrocomia aculeata), guavira (Campomanesia pubescens) and pequi (Caryocar brasiliense) as substrate at different temperatures (25, 30, and 35ºC) during 168 hrs. Results: Samples were taken every 24 hrs, which resulted in 8-points kinetic experiments to determine microbiological and enzymatic contents. The best substrate for β-glucosidase activity was pequi waste after 48 hrs at 30ºC (0.061 U/mL). For amylase activity, bocaiuva presented itself as the best substrate after 96 hrs at 30ºC (0.925 U/mL). CMCase activity was higher in guavira waste after 96 hrs at 35ºC (0.787 U/mL). However, the activity was more expressive for xylanase in substrate composed of bocaiuva residue after 144 hrs at 35ºC (1.802 U/mL). Conclusions: It was concluded that best growth condition for L. ramosa is at 35ºC for all substrates and that xylanase is the enzyme with more potential in SSB, considering the studied Brazilian savannah fruit wastes.