Identifying genes that impact on aroma profiles produced by Saccharomyces cerevisiae and the production of higher alcohols

CITATION: Styger, G., Jacobson, D. & Bauer, F. 2011. Identifying genes that impact on aroma profiles produced by Saccharomyces cerevisiae and the production of higher alcohol. Applied Microbiology and Biotechnology, 91(3):713-30, doi:10.1007/s00253-011-3237-z.The original publication is available at http://www.springerlink.com/content/526367083qt666t3/During alcoholic fermentation, many volatile aroma compounds are formed by Saccharomyces cerevisiae, including esters, fatty acids, and higher alcohols. While the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the regulation of the network, and the physiological roles of individual pathways within the network. Furthermore, different yeast strains tend to produce significantly different aroma profiles. These differences are of tremendous biotechnological interest, since producers of alcoholic beverages such as wine and beer are searching for means to diversify and improve their product range. Various factors such as the redox, energy, and nutritional balance of a cell have previously been suggested to directly or indirectly affect and regulate the network. To gain a better understanding of the regulations and physiological role of this network, we screened a subset of the EUROSCARF strain deletion library for genes that, when deleted, would impact most significantly on the aroma profile produced under fermentative conditions. The 10 genes whose deletion impacted most significantly on higher alcohol production were selected and further characterized to assess their mode of action within or on this metabolic network. This is the first description of a large-scale screening approach using aroma production as the primary selection criteria, and the data suggest that many of the identified genes indeed play central and direct roles within the aroma production network of S. cerevisiae.Publishers' VersionPost prin

Sutherlandia frutescens: The meeting of science and traditional knowledge

Sutherlandia frutescens (L.) R.Br. (syn. Lessertia frutescens (L.) Goldblatt and J.C. Manning) is an indigenous medicinal plant extensively used in South Africa to treat a variety of health conditions. It is a fairly widespread, drought-resistant plant that grows in the Western, Eastern, and Northern Cape provinces and some areas of KwaZulu-Natal, varying in its chemical and genetic makeup across these geographic areas.1 Sutherlandia is widely used as a traditional medicine. Extensive scientific studies are being carried out on the safety, quality, and the efficacy of this medicinal plant to validate the traditional claims, elucidate the bioactive constituents, and conduct clinical trials. This has resulted in a unique situation in South Africa’s history, where traditional knowledge and science intersect to provide insight into this popular plant. This photoessay attempts to illustrate the interlinkage of science with the indigenous knowledge of traditional healers, the local knowledge of people who care for the sick, product development, and innovation agenda of the country as it relates to this plant.Web of Scienc

The role of steroidogenic factor-1 (SF-1) in transcriptional regulation of the gonadotropin-releasing hormone (GnRH) receptor gene

Thesis (MSc)--Stellenbosch University, 2001.ENGLISH ABSTRACT: The GnRH receptor is a G-protein-coupled receptor in pituitary gonadotrope cells. Binding of its ligand, GnRH, results in synthesis and release of gonadotropin hormones luteinizing hormone (LH) and follicle stimulating hormone (FSH). Steroidogenic factor 1 (SF-1), a transcription factor, binds to specific sites in the promoter region of gonadotropin genes, and thus regulates transcription of these genes. The promoter region of the GnRHreceptor gene contains two SF-1-like binding sites, one at -14 to -8 (site 1) and another at -247 to -239 (site 2), relative to the methionine start codon. The role played by these two SF-1-like sites in basal transcription of the mouse GnRH receptor (mGnRH-R) gene in a pituitary precursor gonadotrope cell line, aT3 cells, was the first area of investigation during this study. Luciferase reporter constructs containing 580 bp of mGnRH-R gene promoter were prepared, where SF-1-like sites were either wildtype or mutated. Four such constructs were made, i.e. wildtype (LG), site 1 mutant (LGM1), site 2 mutant (LGM2) and mutated site 1 plus site 2 (LGM1/2). These constructs were transfected into aT3 cells to determine the effect of mutations of sites 1 and/or 2 on the basal expression of the mGnRH-R gene. Mutation of either site 1 or site 2 had no effect on basal expression of the mGnRH-R gene. It was found that only upon simultaneous mutation of both sites 1 and 2, a 50% reduction in basal transcription took place. The implications of this is that SF-1 protein seems to only require one intact DNA-binding site, to mediate basal transcription of the mGnRH-R gene, suggesting that these two sites lie in close proximity during basal transcription. The effect of the protein kinase A (PKA) pathway on the endogenous mGnRH-R gene was also investigated by incubating non- , transfected aT3 cells with the PKA activators, forskolin and 8-Br-cAMP. Similar incubations were also performed on the wild type and mutated site 1 constructs transfected into pituitary gonadotrope aT3 cells. It was found that forskolin and 8-Br-cAMP were able to increase endogenous mGnRH-R mRNA levels in a concentration-dependent fashion, showing that endogenous GnRH receptor gene expression is stimulated via a protein kinase A pathway. Similar results were obtained with the wildtype promoter construct, showing that the protein kinase A pathway stimulates transcription of the promoter. This effect was only seen with wild type and not with the mutated site 1. These results are consistent with a role for a SF-1-like transcription factor in mediating the protein kinase A effect via binding to the site 1 at position -14 in the GnRH receptor gene. A separate investigation was performed to determine whether 25-hydroxycholesterol (25-0HC) is a ligand for SF-1, by incubating aT3 cells transfected with the various constructs with 25-0HC. Results show a dose-dependant response, with an increase in gene expression at 1 μM and a decrease at higher concentrations, for both mutant and wild type constructs. This suggests that, if SF-1 is indeed the protein binding to sites 1 and 2, then 25-0HC is not a ligand for SF-1 protein in aT3 cells and that the effect of 25-0HC on the mGnRH-R gene is not mediated via site 1. The results indicate that these decreases of expression at the higher concentrations may be due to cytotoxic effects. Towards the end of the study the laboratory obtained a luminoskan instrument with automatic dispensing features. Optimisation studies on the luciferase and β-Gal assays were performed on the luminoskan in a bid to decrease experimental error. It was found that automation of these assays resulted in a decrease in experimental error, showing that future researchers could benefit substantially from these optimisation studies.AFRIKAANSE OPSOMMING: Die GnRH reseptor is 'n G proteïen-gekoppelde reseptor in pituitêre gonadotroopselle. Binding van die ligand, GnRH, lei tot die sintese en vrystelling van die gonadotropien hormone, luteïniserende hormoon (LH) en follikel stimulerende hormoon (FSH). Steroidogeniese faktor-t (SF-1) is 'n transkripsie faktor wat aan spesifieke areas in die promotergebied van die gonadotropien hormone bind, en dus transkripsie van hierdie gene reguleer. Die promotergebied van die GnRH reseptor geen bevat twee SF-1 bindings areas, een by -14 to -8 (area 1) asook by -247 to -239 (area 2), relatief to die metionien beginkodon. Die rol wat hierdie twee SF-1 areas speel in basale transkripsie van die muis GnRH reseptor (mGnRH-R) geen in 'n pituïtêre voorloper gonadotroop sellyn, aT3 selle, was die eerste gebied van ondersoek gedurende hierdie studie. Plasmiede bestaande uit die 580 basispaar mGnRH-R promoter verbind aan 'n lusiferase geen is vervaardig, waar SF-1-soortige areas enersyds onveranderd gelaat is, of gemuteer is. Vier sulke plasmiede is vervaardig, nl. onveranderd (LG), area 1 mutant (LGM1), area 2 mutant (LGM2) en gemuteerde area 1 plus area 2 (LGM1/2). Hierdie plasmiede is gebruik om aT3 selle te transfekteer om die effek van mutasies van areas 1 en/of 2 op die basale ekspressie van die mGnRH-R geen te ondersoek. Daar is gevind dat mutasies van areas 1 of 2 geen effek op basale ekspressie op die bogenoemde geen gehad het nie. Slegs tydens gelyktydige mutasie van areas 1 en 2 het 'n 50% vermindering in basale transkripsie plaasgevind. Die implikasies hiervan is dat die SF-1 proteïen blykbaar slegs een volledige DNA-bindingsarea benodig om basale transkripsie van die mGnRH-R geen te reguleer. Dit wil dus voorkom of hierdie twee areas baie na aan mekaar geposisioneer is tydens basale transkripsie. Die effek van die proteïen kinase A (PKA) roete op die natuurlike mGnRH-R geen is ook ondersoek tydens inkubasie van nie-getransfekteerde aT3 selle met die PKA akiveerders, forskolin en 8-Br-cAMP. Soortgelyke inkubasie is ook gedoen op die onveranderde en gemuteerde area 1 plasmiede wat in aT3 selle getransfekteer is. Daar is gevind dat forskolin en 8-Br-cAMP daarin geslaag het om die natuurlike mGnRH-R geen mRNA vlakke op 'n konsentrasie-afhanklike wyse te vermeerder. Hierdie resultaat dui daarop aan dat die natuurlike mGnRH-R geen se ekspressie gestimuleer kan word via 'n proteïen kinase A roete. Soortgelyke resultate is verkry met die onveranderde promoter plasmied en dit wys ook daarop dat proteïen kinase A transkripsie deur die promoter kan stimuleer. Hierdie effek was slegs aanwesig met die onveranderde en nie met die gemuteerde area 1 plasmied nie. Die resultate stem ooreen met 'n rol vir SF-1 transkripsie faktor in die regulering van proteren kinase A effek deur middel van binding aan die area 1 by posisie -14 in die GnRH-R geen. 'n Afsonderlike ondersoek is gedoen om vas te stel of 25-hidroksiecholesterol (25-0HC) 'n ligand vir SF-1 is deur getransfekteerde aT3 selle met 25-0HC te inkubeer. Resultate toon 'n dosis-afhanklike respons met 'n verhoging in geen ekspressie by 1 μM en 'n verlaging met hoër konsentrasies vir beide onveranderde en gemuteerde plasmiede. Dit impliseer dat, indien SF-1 wel die faktor is wat aan areas 1 en 2 bind, 25-0HC nie die ligand vir SF-1 proteren in aT3 selle is nie en dat die effek van 25-0HC op die mGnRH-R geen nie gereguleer word via area 1 nie. Die verlaging in ekspressie gevind by die hoër konsentrasies is dalk die gevolg van sitotoksiese effekte. Teen die einde van die studie het die laboratorium luminoskan toerusting met outomatiese pipettering verkry. Optimiseringstudies van die lusifirase en β-Galtoetse is met die luminoskan gedoen in 'n poging om eksperimentele foute te minimaliseer. Daar is gevind dat outomatisering van hierdie toetse wel gelei het tot 'n verlaging in eksperimentele foute. Toekomstige navorsers kan dus grootliks voordeel trek uit hierdie optimiseringstudies

Elucidating the metabolic pathways responsible for higher alcohol production in Saccharomyces cerevisiae

Thesis (PhD (Wine Biotechnology))--University of Stellenbosch, 2011.Includes bibliography.ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, this study took two approaches: In a first part, the initial aim had been to compare two techniques of transcriptome analysis, DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, while the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional genomics approach by screening a subset of the EUROSCARF strain deletion library, and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway.AFRIKAANSE OPSOMMING: Alkoholiese fermentasie, en veral die maak van wyn, is een van die vroegste mikrobiologiese prosesse wat deur die mensdom ingespan is. Die gisspesie Saccharomyces cerevisiae is gewoonlik grotendeels verantwoordelik vir die fermentasie and verskeie vorige studies het gedemonstreer dat hierdie spesie ‘n baie belangrike rol speel in die uiteindelike kwaliteit en karakter van die voltooide produk. Nieteenstaande die feit is daar steeds baie aspekte van beide die genetiese en metaboliese prosesse wat plaasvind tydens alkoholiese fermentatsie wat nog swak verstaan word, insluitende metaboliese padweë wat ‘n impak het op die smaak en aroma van die fermentasie produk. Om ons kennis van die veld uit te brei het die studie twee aanslae geneem: In die eerste geval is gepoog om twee tegnieke van transkriptoom analiese, nl. DNA oligomikro- arrays en Serial Analysis of Gene Expression (SAGE) te bestudeer vir hul vermoë om geen ekspressie veranderinge tydens wynfermentasie te ondersoek en meer spesifiek om hul potensiaal om ‘n kombinasie van kwantitatiewe sowel as kwalitatiewe data met betreking to mRNA vlakke te produseer. Die SAGE metode kon egter geen betroubare resultate produseer nie en dus word slegs die resultate van die mikro-array eksperimente in die tesis bespreek. Die resultaat is ‘n geheeloorsig oor die geenekspressie veranderinge wat so ‘n wyngis tydens alkoholiese fermentasie ondergaan en dien as ‘n verwysingsraamwerk vir toekomstige studies met geneties gemodifiseerde gis of selfs verskillende fermentasieparameters. Die tweede deel van die studie het gefokus op die identifikasie van gene wat ‘n impak het op die vorming van belangrike, vlugtige aroma komponente, o. a. Esters vetsure en hoër alkohole d.m.v. ‘n siftingseksperiment. Alhoewel daar redelik baie inligting is oor die onderligende metaboliese netwerke wat lei tot die vorming van die verbindings, is daar min kennis van die genetiese regulasie van die netwerk en die fisiologiese rol van individuele padweë wat die netwerk vorm. Verskeie faktore – wat of die netwerk direk of indirek affekteer – is al voorgestel, meer met min konkrete bewyse. Dus het ons gepoog om meer lig op die onderwerp te laat m.b.v. ‘n funksionele genoom aanslag deur ‘n siftingseksperiment te doen op ‘n subgroep (spesifiek gene wat kodeer vir dekarboksilase, dehidrogenase en reduktase ensieme) van die EUROSCARF delesiebiblioteek. Dus is tien gene geïdentifiseer – die delesie waarvan ‘n merkbare effek het op die aroma produksie netwerk en spesifiek die van hoër alkohole. Ooruitdrukkings en enkel en meervoudige delesie rasse van die tien gene is gemaak om d.mv. genetiese analiese, hulle rol in aroma produksie en die Ehrlich padweh uit te pluis. Die resultate toon dat hierdie padweg sensitief is teenoor die sellulêre redoks balans en dui op direkte rolle vir Thi3p, Aad6p en Hom2p, asook dat Bat2p ‘n baie belangrike rol speel in die werking van die padweg

The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates

Copyright © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reservedMariska Lilly, Florian F. Bauer, Gustav Styger, Marius G. Lambrechts & Isak S. Pretoriu