Engineering xylose fermentation in an industrial yeast: continuous cultivation as a tool for selecting improved strains.

Production of second-generation ethanol from lignocellulosic residues should be fueling the energy matrix in the near future. Lignocellulosic biomass has received considerable attention as an alternative renewable resource toward reducing the demand for fossil energy sources, contributing to a future sustainable bio-based economy. Fermentation of lignocellulosic hydrolysates poses many scientific and technological challenges as the drawback of Saccharomyces cerevisiae's inability in fermenting pentose sugars (derived from hemicellulose). To overcome the inability of S. cerevisiae to ferment xylose and increase yeast robustness in the presence of inhibitory compound-containing media, the industrial S. cerevisiae strain SA-1 was engineered using CRISPR-Cas9 with the oxidoreductive xylose pathway from Scheffersomyces stipitis (encoded by XYL1, XYL2, and XYL3). The engineered strain was then cultivated in a xylose-limited chemostat under increasing dilution rates (for 64 days) to improve its xylose consumption kinetics under aerobic conditions. The evolved strain (DPY06) and its parental strain (SA-1 XR/XDH) were evaluated under microaerobic in a hemicellulosic hydrolysate-based medium. DPY06 exhibited 35% higher volumetric ethanol productivity compared to its parental strain.

Quantitative physiology and elemental composition of Kluyveromyces lactis CBS 2359 during growth on glucose at different specific growth rates.

The yeast Kluyveromyces lactis has received attention both from academia and industry due to some important features, such as its capacity to grow in lactose-based media, its safe status, its suitability for large-scale cultivation and for heterologous protein synthesis. It has also been considered as a model organism for genomics and metabolic regulation. Despite this, very few studies were carried out hitherto under strictly controlled conditions, such as those found in a chemostat. Here we report a set of quantitative physiological data generated during chemostat cultivations with the K. lactis CBS 2359 strain, obtained under glucose-limiting and fully aerobic conditions. This dataset serves [corrected] as a basis for the comparison of K. lactis with the model yeast Saccharomyces cerevisiae in terms of their elemental compositions, as well as for future metabolic flux analysis and metabolic modelling studies with K. lactis.

Erratum to: Quantitative physiology and elemental composition of Kluyveromyces lactis CBS 2359 during growth on glucose at different specific growth rates.

In the original publication of the article, the below mentioned errors have appeared. The correct text is provided in this erratum, In the abstract section, the sentence "This dataset serve" should be replaced as "This dataset serves". Also, the reference "Basso TO, Gomes FS, Lopes ML, et al (2014) Homo- and heterofermentative lactobacilli differently affect sugarcane-based fuel ethanol fermentation. Antonie Van Leeuwenhoek 105:169-177. doi: 10.1007/s10482-013-0063-6 " should be replaced as "Basso TO, Dario MG, Tonso A, Stambuk BU, Gombert AK (2010) Insufficient uracil supply in fully aerobic chemostat cultures of Saccharomyces cerevisiae leads to respiro-fermentative metabolism and double nutrient-limitation. Biotechnol Lett 32:973-977. doi: 10.1007/s10529-010-0248-2 ". Finally, in the Table 2 footnote, "according to (Heijnen 1981)" should be replaced as "according to Heijnen (1981)".

Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield.

Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

Mitigating stress in industrial yeasts.

The yeast, Saccharomyces cerevisiae, is the premier fungal cell factory exploited in industrial biotechnology. In particular, ethanol production by yeast fermentation represents the world's foremost biotechnological process, with beverage and fuel ethanol contributing significantly to many countries economic and energy sustainability. During industrial fermentation processes, yeast cells are subjected to several physical, chemical and biological stress factors that can detrimentally affect ethanol yields and overall production efficiency. These stresses include ethanol toxicity, osmostress, nutrient starvation, pH and temperature shock, as well as biotic stress due to contaminating microorganisms. Several cell physiological and genetic approaches to mitigate yeast stress during industrial fermentations can be undertaken, and such approaches will be discussed with reference to stress mitigation in yeasts employed in Brazilian bioethanol processes. This article will highlight the importance of furthering our understanding of key aspects of yeast stress physiology and the beneficial impact this can have more generally on enhancing industrial fungal bioprocesses.

Physiological characterization of a new thermotolerant yeast strain isolated during Brazilian ethanol production, and its application in high-temperature fermentation.

BACKGROUND: The use of thermotolerant yeast strains can improve the efficiency of ethanol fermentation, allowing fermentation to occur at temperatures higher than 40 °C. This characteristic could benefit traditional bio-ethanol production and allow simultaneous saccharification and fermentation (SSF) of starch or lignocellulosic biomass. RESULTS: We identified and characterized the physiology of a new thermotolerant strain (LBGA-01) able to ferment at 40 °C, which is more resistant to stressors as sucrose, furfural and ethanol than CAT-1 industrial strain. Furthermore, this strain showed similar CAT-1 resistance to acetic acid and lactic acid, and it was also able to change the pattern of genes involved in sucrose assimilation (SUC2 and AGT1). Genes related to the production of proteins involved in secondary products of fermentation were also differentially regulated at 40 °C, with reduced expression of genes involved in the formation of glycerol (GPD2), acetate (ALD6 and ALD4), and acetyl-coenzyme A synthetase 2 (ACS2). Fermentation tests using chemostats showed that LBGA-01 had an excellent performance in ethanol production in high temperature. CONCLUSION: The thermotolerant LBGA-01 strain modulates the production of key genes, changing metabolic pathways during high-temperature fermentation, and increasing its resistance to high concentration of ethanol, sugar, lactic acid, acetic acid, and furfural. Results indicate that this strain can be used to improve first- and second-generation ethanol production in Brazil.

The Third International Symposium on Fungal Stress - ISFUS.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

Vinasse fertirrigation alters soil resistome dynamics: an analysis based on metagenomic profiles.

Every year around 300 Gl of vinasse, a by-product of ethanol distillation in sugarcane mills, are flushed into more than 9 Mha of sugarcane cropland in Brazil. This practice links fermentation waste management to fertilization for plant biomass production, and it is known as fertirrigation. Here we evaluate public datasets of soil metagenomes mining for changes in antibiotic resistance genes (ARGs) of soils from sugarcane mesocosms repeatedly amended with vinasse. The metagenomes were annotated using the ResFam database. We found that the abundance of open read frames (ORFs) annotated as ARGs changed significantly across 43 different families (p-value < 0.05). Co-occurrence network analysis revealed distinct patterns of interactions among ARGs, suggesting that nutrient amendment to soil microbial communities can impact on the coevolutionary dynamics of indigenous ARGs within soil resistome.

Calorie restriction hysteretically primes aging Saccharomyces cerevisiae toward more effective oxidative metabolism.

Calorie restriction (CR) is an intervention known to extend the lifespan of a wide variety of organisms. In S. cerevisiae, chronological lifespan is prolonged by decreasing glucose availability in the culture media, a model for CR. The mechanism has been proposed to involve an increase in the oxidative (versus fermentative) metabolism of glucose. Here, we measured wild-type and respiratory incompetent (ρ(0)) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR. The concomitant and quantitative measurements allowed for calculations of conversion factors between different pairs of substrates and products, maximum specific substrate consumption and product formation rates and maximum specific growth rates. Interestingly, we found that the limitation of glucose availability in CR S. cerevisiae cultures hysteretically increases oxygen consumption rates many hours after the complete exhaustion of glucose from the media. Surprisingly, glucose-to-ethanol conversion and cellular growth supported by glucose were not quantitatively altered by CR. Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol. Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ(0) cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.