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BackgroundEscherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose.ResultsThe effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness.ConclusionsCWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.

Lo sviluppo di “fabbriche cellulari” microbiche per la biosintesi di molecole di interesse è uno dei possibili approcci per affrontare l’aumento esponenziale della popolazione. Attualmente in letteratura si ritrovano innumerevoli esempi di questo approccio applicati a svariati prodotti. Trarre profitto dai percorsi metabolici noti è spesso possibile solo attraverso tecniche di ingegneria metabolica, per reindirizzare il metabolismo e forzarlo verso l’accumulo dei metaboliti di interesse. Tra tutti i microorganismi, il lievito Saccharomyces cerevisiae è una delle migliori fabbriche cellulari attualmente utilizzate in numerosi processi industriali, come ad esempio la produzione di biocarburanti. Il corpo principale di questo lavoro è incentrato sull'estensione della gamma di applicazioni per produrre molecole di forte interesse industriale sfruttando le capacità innate e non ancora esplorate di S. cerevisiae. Nella prima parte del lavoro abbiamo valutato la possibilità di produrre ammoniaca. Attualmente, la produzione avviene prevalentemente per sintesi chimica ma la necessità di ridurre l’inquinamento e trovare vie sintetiche rinnovabili ha condotto i ricercatori a testare le produzioni per via microbica. Ispirati dai precedenti studi con batteri, abbiamo indotto S. cerevisiae a sfruttare sostanze azotate naturali attraverso una strategia di evoluzione adattiva. I risultati hanno mostrato una marcata capacità di secernere ammoniaca utilizzando nutrienti non convenzionali. Mediante tecniche di coltivazione in bioreattore e sfruttando il metabolismo naturale del lievito, abbiamo raggiunto 2,03 grammi di ammoniaca totale prodotta che, ad oggi, è il più alto valore registrato con S. cerevisiae. Nella seconda parte di questo lavoro, lo scopo principale è stato quello di esaminare l'accumulo di un altro metabolita chiamato trealosio, esplorando le sue potenziali applicazioni. Abbiamo isolato un ceppo di lievito specifico in cui i geni responsabili della degradaz, The construction of microbial cell factories for the biosynthesis of molecules of interest is one of the essential prerogatives to face an exponential population increase. Taking full advantage of metabolism remains a challenge that has become extremely popular among researchers. Exploiting metabolic pathways using a rewarding approach is often only feasible through metabolic engineering techniques, which enable distinctive utilization of the route to produce compounds of interest. Among all, Saccharomyces cerevisiae is one of the best cell factories currently used in numerous industrial processes, such as the production of biofuels. The main body of this thesis focuses on extending the range of applications to produce molecules of strong industrial interest by exploiting the innate and hidden capabilities of S. cerevisiae. In the first part of this work, we explored the potential to produce ammonia. Currently, production is predominantly by chemical synthesis, and the need to move towards more sustainable society has led researchers to find microorganisms capable of producing chemicals. Intrigued by what had been done in previous studies with bacteria, we induce S. cerevisiae to take advantage of natural nitrogen substrates through an Adaptive Laboratory Evolution strategy. Results showed a marked ability to secrete ammonia revealing a pattern in the use of unconventional substrates. Using bioreactor cultivation techniques and exploiting the unknown natural metabolism of yeast, we achieved 2,03 grams of ammonia, which is the highest recorded with Saccharomyces cerevisiae to date. In the second part, the main purpose was to examine trehalose accumulation and explore its potential applicability. We isolated a specific yeast strain in which the genes responsible for the breakdown of trehalose were deleted (neutral and acid trehalose) and unexpectedly noticed an increased amount of this molecule being mobilized from the cytosol to the extracellular environment. The stu

La relazione negoziale, per sua natura, tende a una infinitezza per le conseguenze che ha sul contesto in cui si sviluppa. Proprio per questo il negoziatore è "colui che cerca la pace, venendo inserito, nonostante i pregiudizi, tra gli ultimi idealisti rimasti". Ciò perché non potrebbe svolgere il proprio lavoro senza credere fermamente nella concordia. Partendo da questo approccio il volume offre una visione originale delle strategie negoziali grazie a un dialogo tra Luca Brambilla, tra i massimi esperti italiani di comunicazione strategica, e Carlo Galimberti, Ordinario di Psicologia sociale della comunicazione presso l'Università Cattolica del Sacro Cuore di Milano.

In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag- phenotype). Rag- mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag- phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2 strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.

Yeast chronological aging is regarded as a model for aging of mammalian post-mitotic cells. It refers to changes occurring in stationary phase cells over a relatively long period of time. How long these cells can survive in such a non-dividing state defines the chronological lifespan. Several factors influence cell survival including two well known normal by-products of yeast glucose fermentation such as ethanol and acetic acid. In fact, the presence in the growth medium of these C2 compounds has been shown to limit the chronological lifespan. In the chronological aging paradigm, a pro-aging role has also emerged for the deacetylase Sir2, the founding member of the Sirtuin family, whose loss of function increases the depletion of extracellular ethanol by an unknown mechanism. Here, we show that lack of Sir2 strongly influences carbon metabolism. In particular, we point out a more efficient acetate utilization which in turn may have a stimulatory effect on ethanol catabolism. This correlates with an enhanced glyoxylate/gluconeogenic flux which is fuelled by the acetyl-CoA produced from the acetate activation. Thus, when growth relies on a respiratory metabolism such as that on ethanol or acetate, SIR2 inactivation favors growth. Moreover, in the chronological aging paradigm, the increase in the acetate metabolism implies that sir2 Delta cells avoid acetic acid accumulation in the medium and deplete ethanol faster; consequently pro-aging extracellular signals are reduced. In addition, an enhanced gluconeogenesis allows replenishment of intracellular glucose stores which may be useful for better long-term cell survival.

A systematic investigation on the effects of auxotrophies on the performance of yeast in aerated fed-batch reactor was carried out. Six isogenic strains from the CEN.PK family of Saccharomyces cerevisiae, one prototroph and five auxotrophs, were grown in aerated fed-batch reactor using the same operative conditions and a proper nutritional supplementation. The performance of the strains, in terms of final biomass decreased with increasing the number of auxotrophies. Auxotrophy for leucine exerted a profound negative effect on the performance of the strains. Accumulation of reactive oxygen species (ROS) in the cells of the strain carrying four auxotrophies and its significant viability loss, were indicative of an oxidative stress response induced by exposure of cells to the environmental conditions. The mathematical model was fundamental to highlight how the carbon flux, depending on the number and type of auxotrophies, was diverted towards the production of increasingly large quantities of energy for maintenance. © 2011 Elsevier Inc.

The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality. © 2011, American Society for Microbiology.

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in expo- nential growing batch cultures at faster growth rate, while localization of catalytic and regulatory sub- units is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol- grown cells appear to depend on both increase in molecule number and kcat. Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism