BackgroundMicrobial population dynamics in bioreactors depend on both nutrients availability and changes in the growth environment. Research is still ongoing on the optimization of bioreactor yields focusing on the increase of the maximum achievable cell density.ResultsA new process-based model is proposed to describe the aerobic growth of Saccharomyces cerevisiae cultured on glucose as carbon and energy source. The model considers the main metabolic routes of glucose assimilation (fermentation to ethanol and respiration) and the occurrence of inhibition due to the accumulation of both ethanol and other self-produced toxic compounds in the medium. Model simulations reproduced data from classic and new experiments of yeast growth in batch and fed-batch cultures. Model and experimental results showed that the growth decline observed in prolonged fed-batch cultures had to be ascribed to self-produced inhibitory compounds other than ethanol.ConclusionsThe presented results clarify the dynamics of microbial growth under different feeding conditions and highlight the relevance of the negative feedback by self-produced inhibitory compounds on the maximum cell densities achieved in a bioreactor.

The mechanisms behind the Warburg effect in mammalian cells, as well as for the similar Crabtree effect in the yeast Saccharomyces cerevisiae, are still a matter of debate: why do cells shift from the energy-efficient respiration to the energy-inefficient fermentation at high sugar concentration?This review reports on the strong similarities of these phenomena in both cell types, discusses the current ideas, and provides a novel interpretation of their common functional mechanism in a dynamic perspective. This is achieved by analysing another phenomenon, the sugar-induced-cell-death (SICD) occurring in yeast at high sugar concentration, to highlight the link between ATP depletion and cell death.The integration between SICD and the dynamic functioning of the glycolytic process, suggests that the Crabtree/Warburg effect may be interpreted as the avoidance of ATP depletion in those conditions where glucose uptake is higher than the downstream processing capability of the second phase of glycolysis. It follows that the down-regulation of respiration is strategic for cell survival allowing the allocation of more resources to the fermentation pathway, thus maintaining the cell energetic homeostasis.

The gene xynC encoding xylanase C from Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 3538 bp DNA fragment containing xynC gene was determined, revealing an open reading frame of 3258 bp that encodes a protein of 120567 Da. A comparison of the deduced amino acid sequence of xylanase C with known /I-glycanase sequences showed that the encoded enzyme is a modular protein containing three different domains. The central region of the enzyme is the catalytic domain, which shows high homology t o family 10 xylanases. A domain homologous t o family IX cellulose-binding domains is located in the C-terminal region of xylanase C, whilst the N-terminal region of the enzyme shows homology t o thermostabilizing domains found in several thermophilic enzymes. Xylanase C showed an activity profile similar t o that of enzymes from mesophilic microorganisms. Maximum activity was found at 45OC, and the enzyme was only stable at 55 O C or lower temperatures. Xylotetraose, xylotriose, xylobiose and xylose were the main products from birchwood xylan hydrolysis, whilst the enzyme showed increasing activity on xylo-oligosaccharides of increasing length, indicating that the cloned enzyme is an endoxylanase. A deletion derivative of xylanase C, lacking the region homologous t o thermostabilizing domains, was constructed. The truncated enzyme showed a lower optimum temperature for activity than the full-length enzyme, 35 O C instead of 4 5 OC, and a reduced thermal stability that resulted in a complete inactivation of the enzyme after 2 h incubation at 55 OC.

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Xylanase A from Bacillus sp. BP7, an enzyme with potential applications in biotechnology, was used to test Pir4, a disulfide bound cell wall protein, as a fusion partner for the expression of recombinant proteins in standard or glycosylation-deficient mnn9 strains of Saccharomyces cerevisiae. Five different constructions were carried out, inserting in-frame the coding sequence of xynA gene in that of PIR4, with or without the loss of specific regions of PIR4. Targeting of the xylanase fusion protein to the cell wall was achieved in two of the five constructions, while secretion to the growth medium was the fate of the gene product of one of the constructions. In all three cases localization of the xylanase fusion proteins was confirmed both by Western blot and detection with Pir-specific antibodies and by xylanase activity determination. The cell wall-targeted fusion proteins could be extracted by reducing agents, showing that the inclusion of a recombinant protein of moderate size does not affect the way Pir4 is attached to the cell wall. Also, the construction that leads to the secretion of the fusion protein permitted us to identify a region of Pir4 responsible for cell wall retention. In summary, we have developed a Pir4-based system that allows selective targeting of an active recombinant enzyme to the cell wall or the growth medium. This system may be of general application for the expression of heterologous proteins in S. cerevisiae for surface display and secretion.

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