Identification of genes affecting glucose catabolism in nitrogen-limited fermentation By Jennifer Margaret Gardner A thesis submitted for the degree of l)octor of Philosophy' in the Faculty of Sciences School of Agriculture and'lVine The University of Adelaide Australia December 2005 THE UNIVERSITY OF ADELAIDE AUSTBALIA C¡UCE Thesìs SummørY Thesis SummarY such as when assimilable nitrogen becomes limiting in fermentation processes winemaking, sugar transport systems of the inoculated yeast are inactivated may and biomass formation is restricted. As a consequence such fermentations at fail to catabolise all available sugar leaving the product out of specification' rectify' greaterrisk of spoilage and deterioration and needing greater input to In recognition of this critical importance of assimilable nitrogen in the has sought successful completion of several fermentation processes, this study group more to develop yeast strains that utilise this typically limited nutrient efficiently. wine strains, usually of saccharomyces cerevisiae, are known to so- differ in the efficiency with which they exploit nitrogen' As a consequence? called 'nitrogen efficient' strains may offer greater prospects for reliable completion of fermentation. with the aid of transposon mutagenesis together with a high throughput method for analysis of multiple micro-fermentations, nitrogen efficient given mutants were identified that were able to catabolise more sugar for a efficiency amount of utilised nitrogen. Mutants displaying improved nitrogen genes were further characterised in shake-flask fermentations and the affected were identified with the assistance of Inverse-PCR' As wine and laboratory yeast strains can be phenotypically different, especially in terms of their ability to affect oenological fermentations, a haploid be derivative of the wine yeast strain L-2056 was developed, such that it could easily geneticallY maniPulated. Of the identified genes, disruption of NG¡R1 and GIDT,lead to an enhanced of a catabolism of sugar in both a laboratory strain and a haploid derivative wine strain of Saccharomyces cerevisiae, during growth in a chemically of NGrRl ot def,rned grape juice medium with limiting nitrogen. Deletion GIDT metabolites also resulted in minor changes to the amounts in which selected Thesis SummarY were produced (determined by HPLC). Biomass yield (measured as dry weight) was also decreased in NGRI mutants' previous studies have demonstrated a strong link between assimilable nitrogen nitrogen and fermentation rate, when other nutrients are not limiting. The total utilised and the timing of nitrogen uptake of ngrll and gid7l strains was found to be very similar to the parent strain. Thus it was hypothesised that differently to ngrll and gidLl strains could be using the available nitrogen enable enhanced glucose catabolism. Deletion of either NGRI or GIDT was found to affect the expression of genes involved in the core pathway for the utilisation of non-preferred nitrogen sources, known as Central Nitrogen Metabolism (CNM)' The transcriptional GLNL abundance, measured by Real-Time PcR, of GDHI, GDH2, GLTI and genes could was altered in these mutants. This distorted expression of CNM of the translate to a re-modelling of enzyme quantities and thus re-distribution core nitrogen-containing compounds, and thereby the cellular response under nitrogen-limiting conditions' ll Declarøtion Declaration of AuthorshiP of any This thesis contains no material which has been accepted for the award and' to the other degree or diploma in any university or other l.rofüary institution or written by best of my knowledge, contains no material previously published another person, except where reference has been made in the text' in Essentially all of the work detailed in chapters 3 and 4 has been published And the the form of two scientific articles. These are included in Appendix II' references are: Gardner, J. M., McBryde, C. M., Vystavelovã, Ã',de Barros Lopes' M'' Jiranek,V. (2005). Identification of genes affecting glucose catabolism in nitrogen-1i-ìt"d fermentatio n. F E MS Yeast Res earch 5, 79 1 -800. Walker, M. 8., Gardner, J. M., Vystavelovâ, A', McBryde, C', de Barros Lopes, tVr. an¿ Jiranek, v. (2003). Application of th-" reuseable, Kanltlx selåctable marker to industrial yeast: construction and evaluation of heterothallic wine strains of Saccharomyces cerevisiae, possessing minimal foreign DNA sequences. FEMS Yeast Research 41339-347 ' This thesis may be made available for loan or photocopying' Jennifer M. Gardner December 2005 lll Acknowledgements Acknowledgements for First of all, my sincere thanks to my principle supervisor Vladimir Jiranek It has his tremendous support, enthusiasm and patience through out this study. Miguel de been a pleasure to learn from you. Also, thanks to my co-supervisor Barros Lopes, for sharing his knowledge and for help with many technical aspects of this studY. Thank you to the past and present members of the Microbial Biotechnology In Laboratory, it has been a joy to work alongside such talented people' for particular, Michelle Walker, for the keen sharing of ideas and Kate Poole your waÍn friendship, laughter and all the beer' I would like to thank my family, my parents, Elaine and Will, for their never- sister, sonia ending love and encouragement, my brother, Andrew and adopted for their joyousness and my aunt and uncle, Jennifer and Douglas for our friendship, lively discussions and all the magnificent food and wine. Finally, I would like to thank Colin for sharing all the things we love and his courageous persistence that have made our dreams a reality. This project was supported financially by Australia's grapegrowers and winemakers through their investment body the Grape and Wine Research and Development corporation (GWRDC). Further support was kindly provided by Lallemand Australia. I would also like to thank the Australian Postgraduate Award Programme and the GWRDC for the generous scholarship. 1V Abbrevíøtions Abbreviations A adenine ABP anchor bubble Primer AP1 activating protein 1 ARE activating protein I responsive element bp base pair OC degrees centrigrade C cytosine cAMP cyclic AMP CDGJM chemically defined grape juice medium CER carbon dioxide evolution rate cm centimetre CNM central nitrogen metabolism COz carbon dioxide Cr crossing threshold DAP diammonium PhosPhate DIG digoxigenin-l1-dUTP DNA deoxyribonucleic acid dNTPs deoxynucleotide triPhosPhates EMS ethylmethane sulfonate FAN free amino nitrogen G guanosine Þû gram xg x gravity GABA 4-amino butyrate GCN general control of amino acid synthesis GDH glutamate dehYdrogenase GFP green fluorescent Protein GOGAT glutamate sYnthase GS glutamine synthetase h hour HzS hydrogen sulphide HNE high nitro gen efficiencY hnRNPs heterogeneous nuclear ribonuclear proteins HOG high osmolaritY glYcerol HPLC high performance liquid chromatography HSE heat shock elements IMVS Institute for Molecular and Veterinary Science IPCR inverse PCR L litre LB Luria-Bertani M molar MAP mitogen activated Protein min minute mL millilitre mg milligram mM millimolar mol mole mRNA messenger RNA V Abbrevíutions NiMIP asparagine / methionine / Proline NAD+ nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide reduced form NADP nicotinamide adenine dinucleotide phosphate NADPH nicotinamide adenine dinucleotide phosphate reduced form NCR nitrogen catabolite rePression nmol nanomole NOPA opthaldialdehyde/n- acetyl-L - cysteine NRD negative regulatory domain OD optical density PCR polymerase chain reaction PKA protein kinase A pmol picomole reaction QRTPCR quantitative real time polymerase chain RNA ribonucleic acid rpm revolutions per minute RRM RNA recognition motif sec second SDS sodium dodecyl sulPhate snRNPs small nuclear ribonuclear proteins STRE stress responsive element TCA tri-carboxylic acid Tn transposon TNBS ninhydrin, 2,4,6 -tr inrtr ob enzene sul foni c ac i d TOR target of rapamycin U units UAS upstream activating sequence UASNTn nitrogen regulated upstream activating sequence pL microlitre pM micromolar URS upstream regulatiry sequence V volt vlv volume per volume VA volatile acidity w/v weight per volume YAN yeast assimilable nitrogen YEPD yeast extract PePtone dextrose vl Tøhle of Contents Table of Contents I Thesis Summary Declaration of AuthorshiP iii Acknowledgements iv Abbreviations v Chapter 1 Literature Review 1.1 Introduction 1 1.2 Stuck fermentation 2 1.3 The effect of nitrogen on fermentation dynamics 6 1.4 The physiological effects of nitrogen starvation during 8 fermentation 9 1.5 Nitrogen compounds in wine 10 1.6 The nitrogen demand of Yeast 12 1.7 Yeast r"*. nitrogen sources in the extracellular environment 14 1.8 Yeast cells monitõr their intracellular nitrogen pools 15 1.9 Nitrogen catabolite repression 18 1.10 Nitrolen import into ihe cell 20 1.11 Centrãt Nitrãgen Metabolism t.t2 Links between nitrogen and carbon metabolism 2l 22 1.13 The effect of yeast strain and nitrogen utilisation on wine aroma t.l4 Concluding siatement 24 Chapter 2 Materials and Methods 26 2.1 Yeast strains and maintenance 26 2.2 Bacterial strains and maintenance 26 2.3 Culture media 2.3.1 Mediafor yeast cultures 26 2.3.2. Chemically defined grapeiuice media 26 2.4 Growth and fermentation 27 2.4.1 Mini-fermentations 27 2.4.2 Laboratory scale anaerobic fermentations 21 2.4.3 Dry cell weight determination 28 2.4.4 Determination of glucose and ammonia by enzymatic 28 analysis 2.4.5 Determination of other metabolites by HPLC 29 2.4.6 Viable cell counts