BackgroundIt has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes.ResultsOverexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25°C to 20°C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced.ConclusionMonitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.
The kinetics of maltose transport by two industrial yeasts were studied. The ale and lager strain each showed both high and low affinity transport. For the lager strain, maltose transport was only weakly inhibited by maltotriose, sucrose and trehalose, suggesting that its dominant maltose transporter is the maltose-specific type coded by MALx1 genes. For the ale strain, maltose transport was strongly inhibited by maltotriose, sucrose and trehalose, suggesting that its dominant maltose transporter may be the AGT1-encoded type that also carries these sugars. Also glucose inhibited transport by the ale strain more than that by the lager strain. Instantaneous inhibition by ethanol at concentrations met in brewery fermentations was moderate (about 25% at 50 g ethanol · L -1 ). The apparent Vmax for high affinity transport increased about 100-fold between 0 and 30°C, whereas the Km (3 ± 1 mM) was constant. Standard activities of maltose transport and maltase were followed through pilot fermentations of 11-24°P worts. Rapid (20 s) measurements of the zero-trans-rate of maltose uptake were also made with each day's yeast (rapidly harvested and washed) in reaction mixtures containing the same day's wort labelled with tracer 14 C-maltose. Results suggested that maltose uptake is the dominant factor controlling the rate of maltose utilization in these wort fermentations.Key words: Brewing fermentation, catabolite inactivation, glucose repression, maltose transporters, transport inhibition. -2863(9'8-32The main fermentable sugars in brewing fermentation are maltose, glucose and maltotriose. These sugars interfere with each others' catabolism (reviewed by SpencerMartins et al. 23 ). Maltose transporters and ␣-glucosidases (maltases) are encoded, respectively, by MALx1 and MALx2 genes (x = 1-4 and 6, corresponding to different MAL loci, located on different chromosomes and each containing one or more copies of the different MAL genes). MALx3 produces an activator of the other two MAL genes. These genes are repressed by glucose and also by fructose and sucrose 5 . Especially during N-starvation 16 , glucose and maltose itself 9,21 induce catabolite inactivation of maltose transporters but not of maltase 6 . This inactivation involves the proteolysis of already existing transporter molecules. Ethanol is known to inhibit the endocytosis and subsequent proteolysis of maltose transporter, which could play a role in fermentations conditions where the ethanol concentration is high at the end of the process 2,15 .In addition, maltose and maltotriose compete for the same transporter in strains containing the AGT1-coded transporter, which carries several ␣-glucosides 8,24 , whereas glucose and maltose inhibit each other's transport in some strains, although they are not carried by the same transporters 3,23 . Jespersen et al. 11 screened 5 ale yeasts and 25 lager yeasts and found that all contained MALx1 genes and all but one also contained AGT1 genes.It has often been suggested that brewing fermentations could be accelerated if yeast...
BackgroundGrowth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.ResultsWe have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response.ConclusionsBased on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR).
An adaptive evolution method to obtain stable Saccharomyces pastorianus brewing yeast variants with improved fermentation capacity is described. The procedure involved selection for rapid growth resumption at high osmotic strength. It was applied to a lager strain and to a previously isolated ethanol-tolerant strain. Fermentation performance of strains was compared at 15 °P wort strength. A selected osmotolerant variant of the ethanol-tolerant strain showed significantly shorter fermentation time than the parent strain, producing 6.45% alcohol by volume beer in 4-5 days with mostly similar organoleptic properties to the original strain. Diacetyl and pentanedione contents were 50-75% and 3-methylbutyl acetate and 2-phenylethyl acetate 50% higher than with the original strain, leading to a small flavour change. The variant contained significantly less intracellular trehalose and glycogen than the parent. Transcriptional analysis of selected genes at 24 h revealed reduced transcription of hexose transport genes and increased transcription of the MALx1 and MALx2 genes, responsible for α-glucoside uptake and metabolism. It is suggested that an attenuated stress response contributes to the improved fermentation performance. Results show that sequential selection for both ethanol tolerance and rapid growth at high osmotic strength can provide strains with enhanced fermentation speed with acceptable product quality.
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