Prospecting and engineering yeasts for ethanol production under inhibitory conditions: an experimental design analysis.

The recently discovered wild yeast Wickerhamomyces sp. UFFS-CE-3.1.2 was analyzed through a high-throughput experimental design to improve ethanol yields in synthetic media with glucose, xylose, and cellobiose as carbon sources and acetic acid, furfural, formic acid, and NaCl as fermentation inhibitors. After Plackett-Burman (PB) and central composite design (CCD), the optimized condition was used in a fermentation kinetic analysis to compare this yeast's performance with an industrial Saccharomyces cerevisiae strain (JDY-01) genetically engineered to achieve a higher xylose fermentation capacity and fermentation inhibitors tolerance by overexpressing the genes XYL1, XYL2, XKS1, and TAL1. Our results show that furfural and NaCl had no significant effect on sugar consumption by UFFS-CE-3.1.2. Surprisingly, acetic acid negatively affected glucose but not xylose and cellobiose consumption. In contrast, the pH positively affected all the analyzed responses, indicating a cell's preference for alkaline environments. In the CCD, sugar concentration negatively affected the yields of ethanol, xylitol, and cellular biomass. Therefore, fermentation kinetics were carried out with the average concentrations of sugars and fermentation inhibitors and the highest tested pH value (8.0). Although UFFS-CE-3.1.2 fermented glucose efficiently, xylose and cellobiose were mainly used for cellular growth. Interestingly, the genetically engineered strain JDY-01 consumed ~ 30% more xylose and produced ~ 20% more ethanol. Also, while UFFS-CE-3.1.2 only consumed 32% of the acetic acid of the medium, JDY-01 consumed > 60% of it, reducing its toxic effects. Thus, the overexpressed genes played an essential role in the inhibitors' tolerance, and the applied engineering strategy may help improve 2G ethanol production.

Production of ethanol fuel from enzyme-treated sugarcane bagasse hydrolysate using d-xylose-fermenting wild yeast isolated from Brazilian biomes.

In this study, we evaluated the potential of yeasts isolated from Amazon to produce second-generation ethanol from sugarcane bagasse delignified with alkaline hydrogen peroxide and hydrolysed with commercial enzyme preparation. The best efficiency savings in glucose and release of xylose were determined by considering the solids and enzyme loads. Furthermore, we selected Spathaspora passalidarum UFMG-CM-Y473 strain with the best fermentative parameters. Fermentations used bagasse hydrolysate without any nutritional supplementation, a significant difference from previous studies, which is closer to industrial conditions. Ethanol yield of 0.32 g/g and ethanol productivity of 0.34 g/L h were achieved after the consumption of 78% of the sugar. This hydrolysis/fermentation technology package could represent the input of an additional 3180 L of ethanol per hectare in areas of average sugarcane productivity such as 60 ton/ha. Thus, we concluded that Sp. passalidarum UFMG-CM-Y473 has a clear potential for the production of second-generation ethanol from delignified and enzyme-hydrolysed bagasse.

Sucrose and Saccharomyces cerevisiae: a relationship most sweet.

Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering.

What do we know about the yeast strains from the Brazilian fuel ethanol industry?

The production of fuel ethanol from sugarcane-based raw materials in Brazil is a successful example of a large-scale bioprocess that delivers an advanced biofuel at competitive prices and low environmental impact. Two to three fed-batch fermentations per day, with acid treatment of the yeast cream between consecutive cycles, during 6-8 months of uninterrupted production in a nonaseptic environment are some of the features that make the Brazilian process quite peculiar. Along the past decades, some wild Saccharomyces cerevisiae strains were isolated, identified, characterized, and eventually, reintroduced into the process, enabling us to build up knowledge on these organisms. This information, combined with physiological studies in the laboratory and, more recently, genome sequencing data, has allowed us to start clarifying why and how these strains behave differently from the better known laboratory, wine, beer, and baker's strains. All these issues are covered in this minireview, which also presents a brief discussion on future directions in the field and on the perspectives of introducing genetically modified strains in this industrial process.

Insufficient uracil supply in fully aerobic chemostat cultures of Saccharomyces cerevisiae leads to respiro-fermentative metabolism and double nutrient-limitation.

A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h(-1), which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO2 production and O2 consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.

Regulation of Saccharomyces cerevisiae maltose fermentation by cold temperature and CSF1

Foi estudado o efeito da baixa temperatura (10ºC) na fermentação de maltose por uma cepa de S. cerevisiae selvagem, e uma cepa csf1D mutante incapaz de transportar glicose e leucina a baixas temperaturas. A baixa temperatura afeta a cinética da fermentação por diminuir a velocidade de crescimento e rendimento celular final, com quase nenhum etanol produzido a partir de maltose pelas células selvagems a 10ºC. A cepa csf1D foi incapaz de crescer em maltose a 10ºC, indicando que o gene CSF1 é também necessário para a utilização de maltose a baixas temperaturas. Entretanto, o mutante também mostrou inibição acentuada da fermentação de glicose e maltose por estresse salino, indicando que CSF1 também estaria envolvido na regulação de outros processos fisiológicos, incluindo a homeostase iónica.

Regulation of Saccharomyces cerevisiae maltose fermentation by cold temperature and CSF1

We studied the influence of cold temperature (10ºC) on the fermentation of maltose by a S. cerevisiae wild-type strain, and a csf1delta mutant impaired in glucose and leucine uptake at low temperatures. Cold temperature affected the fermentation kinetics by decreasing the growth rate and the final cell yield, with almost no ethanol been produced from maltose by the wild-type cells at 10ºC. The csf1delta strain did not grew on maltose when cultured at 10ºC, indicating that the CSF1 gene is also required for maltose consumption at low temperatures. However, this mutant also showed increased inhibition of glucose and maltose fermentation under salt stress, indicating that CSF1 is probably involved in the regulation of other physiological processes, including ion homeostasis.

Foi estudado o efeito da baixa temperatura (10ºC) na fermentação de maltose por uma cepa de S. cerevisiae selvagem, e uma cepa csf1delta mutante incapaz de transportar glicose e leucina a baixas temperaturas. A baixa temperatura afeta a cinética da fermentação por diminuir a velocidade de crescimento e rendimento celular final, com quase nenhum etanol produzido a partir de maltose pelas células selvagems a 10ºC. A cepa csf1delta foi incapaz de crescer em maltose a 10ºC, indicando que o gene CSF1 é também necessário para a utilização de maltose a baixas temperaturas. Entretanto, o mutante também mostrou inibição acentuada da fermentação de glicose e maltose por estresse salino, indicando que CSF1 também estaria envolvido na regulação de outros processos fisiológicos, incluindo a homeostase iónica.