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Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using “–omics” techniques

ABSTRACT: Addition of sugar syrups to the basic wort is a popular technique to achieve higher gravity in beer fermentations, but it results in dilution of the free amino nitrogen (FAN) content in the medium. The multicomponent protease enzyme Flavourzyme has beneficial effect on the brewer´s yeast fermentation performance during high gravity fermentations as it increases the initial FAN value and results in higher FAN uptake, higher specific growth rate, higher ethanol yield and improved flavour profile. In the present study, transcriptome and metabolome analysis were used to elucidate the effect on the addition of the multicomponent protease enzyme Flavourzyme and its influence on the metabolism of the brewer´s yeast strain Weihenstephan 34/70. The study underlines the importance of sufficient nitrogen availability during the course of beer fermentation. The applied metabolome and transcriptome analysis allowed mapping the effect of the wort sugar composition on the nitrogen uptake. Both the transcriptome and the metabolome analysis revealed that there is a significantly higher impact of protease addition for maltose syrup supplemented fermentations, while addition of glucose syrup to increase the gravity in the wort resulted in increased glucose repression that lead to inhibition of amino acid uptake and hereby inhibited the effect of the protease addition. Keywords: 21 Plato with two carbon sources (glucose and maltose) and with/without enzyme supplementation

ORGANISM(S): Saccharomyces pastorianus Weihenstephan 34/70 Saccharomyces cerevisiae Schizosaccharomyces pombe

PROVIDER: GSE24645 | GEO | 2012/02/12

SECONDARY ACCESSION(S): PRJNA132537

REPOSITORIES: GEO

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Project description:For over 30 years, serine hydroxamate has been used to chemically stimulate a stringent response in Escherichia coli and other bacteria. These studies have elucidated numerous characteristics of the classical stringent response beyond the simple cellular response to an amino acid shortage, including phospholipid synthesis and protease up-regulation. In this study, the effects of a serine hydroxamate addition on high cell density recombinant E. coli were examined and compared to the effects of recombinant protein production to determine overlaps, as recombinant protein production stress has often been attributed to amino acid shortages. Both the transcriptome and growth characteristics were evaluated and compared. The serine hydroxamate addition profoundly decreased the culture growth rate, whereas, recombinant protein production did not. Conversely, the transcriptome profile of the recombinant E. coli cultures were relatively unaffected by the serine hydroxamate addition, yet recombinant protein production dramatically changed the transcriptome profile. A subset of the classical stringent response genes were effected by the serine hydroxamate addition, whereas, recombinant protein production regulated numerous classical stringent response genes; however, not all. The genes that were regulated by the serine hydroxamate addition include numerous fatty acid synthesis genes, in agreement with altered phospholipids synthesis reports. These results indicate that recombinant protein production and the stringent response have many overlapping responses; however, are far from identical. It was hypothesized that recombinant protein production leads to a stringent response due to the high amino acid synthesis demands related to recombinant protein synthesis. A comparison of the transcriptomes during recombinant protein production and a chemical imposed stringent response would assist with determining what portion of the “metabolic burden” associated with recombinant protein production is due to amino acid shortages. In this study, the transcriptome profiles of recombinant E. coli were examined and compared for the three culture conditions: 1) Normal growth, no external stress; 2) L-serine hydroxamate addition (to mediate a stringent response); and 3) IPTG-induction to produce the recombinant protein chloramphenicol acetyltransferase (CAT). The transcriptome profiles from these three conditions were analyzed using Affymetrix Anti-sense E. coli GeneChip® microarrays. Experiment Overall Design: For the serine hydroxamate fermentations, cells were harvested immediately prior to the serine hydroxamate-addition (Time S0) and 1-hour post serine hydroxamate-addition (Time S1). For the IPTG-induced fermentations, cells were harvested immediately prior to the IPTG-addition (Time S0) and 1-hour post-induction (Time S1). For the uninduced (unstressed) fermentations, cells were harvested at Time S0 and Time S1, for which the timing was synchronized with the serine hydroxamate and IPTG fermentations based on OD for Time S0 and time for Time S1. Each fermentation condition was repeated (two biological replicates). RNA from each biological replicates was purified and processed independently. Three biological replicates were obtained for the control condition (Time S0), since prior the serine hydroxamate- or IPTG- addition all fermentations were replicates. Prior to DNA microarray hybridization, where only two biological replicates existed, one of the processed samples was divided into two technical replicates, resulting in three separate hybridized chips. All Time S1 samples contained three technical replicates from two biological duplicates, whereas the control Time S0 measurement contained three biological replicates.

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Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using “–omics” techniques

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