Effect of auxotrophies on yeast performance in aerated fed-batch reactor

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.     

Interleukin-1β production by Zygosaccharomyces bailii [pZ3 KlIL-1β] in aerated fed-batch reactor: Importance of inoculum physiology and bioprocess modelling

A cultural system, aimed at the production of human interleukin-1β (IL-1β) with cells of a non-conventional yeast transformed for interleukin expression, Zygosaccharomyces bailii [pZ₃KlIL-1β], was realized. Interleukin production was accomplished in a reactor operating in fed-batch mode to avoid sugar overflow metabolism, limitations with respect to oxygen transfer, and achieve high cell density. Batch operation mode was employed only to characterise the producer strain and experimentally estimate kinetic parameters. In parallel with strain characterisation, a mathematical model was developed. The comparison between simulations and experimental data allowed to evidence the importance of physiological state of inoculum, being only a fermentative one suitable to sustain a given exponential growth. The respiratory capacity of Z. bailii [pZ₃KlIL-1β], resulted to be affected by stirring. The theoretical and experimental approach allowed the bioprocess optimisation.     

Entrapment of microbial cells within a gelatin matrix: A comparison of three procedures

Summary Three of the methods in which a gelatin matrix is used to entrap microbial cells are compared. The advantages and disadvantages of each method when used for immobilizing invertase-active cells of yeast (Saccharomyces cerevisae) are discussed.     

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Background  

Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain.     

Effect of gelatin-immobilization on the catalytic activity of enzymes and microbial cells

Summary The immobilization of enzymes and microbial cells within insolubilized gelatin involves both physical entrapment and covalent crosslinking, each one playing its role. The effect of this dual type of bonding on the kinetic parameters and activity yield of three enzymes (acid phosphatase, invertase and -glucosidase) and of whole microbial cells belonging to three yeast species (Saccharomyces cerevisiae,Candida utilis andKluyveromyces marxianus) have been investigated.     

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Background

Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae.

Results

To investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H⁺/glycerol transporters.

Conclusion

This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org.     

Immobilization of viable yeast cells within polyaldehyde-hardened gelatin gel

Summary A gel-entrapment method particularly suitable for viable cells is described. The gel matrix is gelatin insolubilized by interaction with polymeric aldehydes (polyaldehydes) prepared by periodate oxidation of polysaccharides such as starch and dextran. The viability of the entrapped cells is evidenced by growth measurement and by SEM analysis of the immobilizate.