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When exploited as cell factories, Saccharomyces cerevisiae cells are exposed to harsh environmental stresses impairing titer, yield and productivity of the fermentative processes. The development of robust strains therefore represents a pivotal challenge for the implementation of cost-effective bioprocesses. Altering master regulators of general cellular rewiring represents a possible strategy to evoke shaded potential that may accomplish the desirable features. The poly(A) binding protein Pab1, as stress granules component, was here selected as the target for obtaining widespread alterations in mRNA metabolism, resulting in stress tolerant phenotypes. Firstly, we demonstrated that the modulation of Pab1 levels improves robustness against different stressors. Secondly, the mutagenesis of PAB1 and the application of a specific screening protocol on acetic acid enriched medium allowed the isolation of the further ameliorated mutant pab1 A60-9. These findings pave the way for a novel approach to unlock industrially promising phenotypes through the modulation of a post-transcriptional regulatory element.

Saccharomyces cerevisiae is one of the most employed cell factories for the production of bioproducts. Although monomeric hexose sugars constitute the preferential carbon source, this yeast can grow on a wide variety of nitrogen sources that are catabolized through central nitrogen metabolism (CNM). To evaluate the effects of internal perturbations on nitrogen utilization, we characterized strains deleted or overexpressed in GLT1, encoding for one of the key enzymes of the CNM node, the glutamate synthase. These strains, together with the parental strain as control, have been cultivated in minimal medium formulated with ammonium sulfate, glutamate, or glutamine as nitrogen source. Growth kinetics, together with the determination of protein content, viability, and reactive oxygen species (ROS) accumulation at the single cell level, revealed that GLT1 modulations do not significantly influence the cellular physiology, whereas the nitrogen source does. As important exceptions, GLT1 deletion negatively affected the scavenging activity of glutamate against ROS accumulation, when cells were treated with H2O2, whereas Glt1p overproduction led to lower viability in glutamine medium. Overall, this confirms the robustness of the CNM node against internal perturbations, but, at the same time, highlights its plasticity in respect to the environment. Considering that side-stream protein-rich waste materials are emerging as substrates to be used in an integrated biorefinery, these results underline the importance of preliminarily evaluating the best nitrogen source not only for media formulation, but also for the overall economics of the process.

In the manufacturing of baker's yeast by aerobic fed-batch systems, continuous assessment of the state of the process is necessary for regulating the flow rate (on/off) for growth medium addition. A new, simple method for the fed-batch yeast process has been developed. It is based on pH changes as a suitable parameter for regulating the feed of fresh concentrated medium in response to metabolic activities of the yeast population. Experimental results have shown that it enables the attaining of high cell density with both high productivity and high yields

Development of robust yeast strains that can efficiently ferment lignocellulose-based feedstocks is one of the requirements for achieving economically feasible bioethanol production processes. With this goal, several genes have been identified as promising candidates to confer improved tolerance to S. cerevisiae. In most of the cases, however, the evaluation of the genetic modification was performed only in laboratory strains, that is, in strains that are known to be quite sensitive to various types of stresses. In the present study, we evaluated the effects of overexpressing genes encoding the transcription factor (YAP1) and the mitochondrial NADH-cytochrome b5 reductase (MCR1), either alone or in combination, in an already robust and xylose-consuming industrial strain of S. cerevisiae and evaluated the effect during the fermentation of undiluted and undetoxified spruce hydrolysate. Overexpression of either gene resulted in faster hexose catabolism, but no cumulative effect was observed with the simultaneous overexpression. The improved phenotype of MCR1 overexpression appeared to be related, at least in part, to a faster furaldehyde reduction capacity, indicating that this reductase may have a wider substrate range than previously reported. Unexpectedly a decreased xylose fermentation rate was also observed in YAP1 overexpressing strains and possible reasons behind this phenotype are discussed.

Microorganisms offer a tremendous potential as cell factories, and they are indeed used by humans for centuries for biotransformations. Among them, yeasts combine the advantage of unicellular state with a eukaryotic organization, and, in the era of biorefi neries, their biodiversity can offer solutions to specifi c process constraints. Zygosaccharomyces bailii, an ascomycetales budding yeast, is widely known for its peculiar tolerance to various stresses, among which are organic acids. Despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here, we describe in detail protocols for transformation, for target gene disruption or gene integration, and for designing episomal expression plasmids helpful for developing and further studying the yeast Z. bailii.

Yeasts play a significant role in biotechnology. They are important production hosts for chemicals, enzymes and biopharmaceutical ingredients. This chapter outlines our current knowledge about the interrelation of the central carbon metabolism and various microbial production processes. It is obvious that every production process is inevitably connected to and dependent of the central carbon metabolism. For primary metabolites these relations are known and described in detail. However our knowledge how central carbon metabolism translates into flux and titer of secondary products and proteins are surprisingly scarce. The connection between central metabolic routes and specific product related pathways are described and metabolic engineering approaches towards enhanced production are discussed.

Microorganisms appear as ideal catalysts for chemical conversions. Diverse metabolic routes seem to open doors to the whole range of chemistry. Indeed, a vast amount of scientific papers suggesting new microbial cell factories for old and new products is published every year. However, only very few of them reached industrial relevance. Chemical balances and some metabolic tricks allow natural microorganisms the efficient production of some chemicals, but not others. So first of all it is important to choose metabolically feasible products of value for synthetic chemistry. Here we see a clear task for the chemical and biotechnology industries to communicate for defining the right target molecules. Finally, despite our limited current knowledge, synthetic biology points to a future independent from natural strain backgrounds

Background: The great interest in the production of highly pure lactic acid enantiomers comes from the application of polylactic acid (PLA) for the production of biodegradable plastics. Yeasts can be considered as alternative cell factories to lactic acid bacteria for lactic acid production, despite not being natural producers, since they can better tolerate acidic environments. We have previously described metabolically engineered Saccharomyces cerevisiae strains producing high amounts of L-lactic acid (>60 g/L) at low pH. The high product concentration represents the major limiting step of the process, mainly because of its toxic effects. Therefore, our goal was the identification of novel targets for strain improvement possibly involved in the yeast response to lactic acid stress. Results: The enzyme S-adenosylmethionine (SAM) synthetase catalyses the only known reaction leading to the biosynthesis of SAM, an important cellular cofactor. SAM is involved in phospholipid biosynthesis and hence in membrane remodelling during acid stress. Since only the enzyme isoform 2 seems to be responsive to membrane related signals (e.g. myo-inositol), Sam2p was tagged with GFP to analyse its abundance and cellular localization under different stress conditions. Western blot analyses showed that lactic acid exposure correlates with an increase in protein levels. The SAM2 gene was then overexpressed and deleted in laboratory strains. Remarkably, in the BY4741 strain its deletion conferred higher resistance to lactic acid, while its overexpression was detrimental. Therefore, SAM2 was deleted in a strain previously engineered and evolved for industrial lactic acid production and tolerance, resulting in higher production. Conclusions: Here we demonstrated that the modulation of SAM2 can have different outcomes, from clear effects to no significant phenotypic responses, upon lactic acid stress in different genetic backgrounds, and that at least in one genetic background SAM2 deletion

Background: Oleaginous microorganisms, such as different yeast and algal species, can represent a sustainable alternative to plant oil for the production of biodiesel. They can accumulate fatty acids (FA) up to 70% of their dry weight with a predominance of (mono)unsaturated species, similarly to what plants do, but differently from animals. In addition, their growth is not in competition either with food, feed crops, or with agricultural land. Despite these advantages, the exploitation of the single cell oil system is still at an early developmental stage. Cultivation mode and conditions, as well as lipid extraction technologies, represent the main limitations. The monitoring of lipid accumulation in oleaginous microorganisms is consequently crucial to develop and validate new approaches, but at present the majority of the available techniques is time consuming, invasive and, when relying on lipid extraction, can be affected by FA degradation. Results: In this work the fatty acid accumulation of the oleaginous yeasts Cryptococcus curvatus and Rhodosporidium toruloides and of the non-oleaginous yeast Saccharomyces cerevisiae (as a negative control) was monitored in situ by Fourier Transform Infrared Spectroscopy (FTIR). Indeed, this spectroscopic tool can provide complementary information to those obtained by classical techniques, such as microscopy, flow cytometry and gas chromatography. As shown in this work, through the analysis of the absorption spectra of intact oleaginous microorganisms it is possible not only to monitor the progression of FA accumulation but also to identify the most represented classes of the produced lipids. Conclusions: Here we propose FTIR microspectroscopy - supported by multivariate analysis - as a fast, reliable and non invasive method to monitor and analyze FA accumulation in intact oleaginous yeasts. The results obtained by the FTIR approach were in agreement with those obtained by the other classical methods like flow cytometry and

Background: The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized.Results: In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes.Conclusions: In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to th

In the context of the global objective of shifting from petroleum to a biomass-based economy, the research on fermentative strategies to produce alternative biofuels and chemicals has become a predominant field of study. Microorganisms, because of their substrate versatility and metabolic efficiency, are promising to partially support our increasing needs for materials and fuels, opening up scenarios for the use of alternative sources, including wastes. Butanol is a very attractive molecule since it can be seen both as a chemical platform and as a fuel. Today, it is principally derived from petroleum, but it also represents the final product of a microbial fermentation. Although Clostridia are the natural and traditional organisms employed in butanol production, systematic approaches to improve production and resistance traits are currently impeded by a lack of characterization and genetic tools. This is the main reason why, besides their optimizations, a significant and growing amount of research is centered on the engineering of alternative robust cell factories capable of elevated production, possibly combined with higher tolerance. Here, we review the most recent advances in n-butanol production in non-Clostridial microbial hosts, including not only other prokaryotic but also eukaryotic microorganisms, which might eventually be seen as second-generation hosts.