The transformation of wine yeasts with glucanase, xylanase and pectinase genes for improved clarification and filterability of wine

Strauss, Marlene (2003-03)

Thesis (MScAgric) -- Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: Cellulose is by far the most abundant carbohydrate available from plant biomass. These biopolymers are therefore an important renewable source of food, fuels and chemicals. Cellulose is embedded in a matrix of hemicellulose, lignin and pectin and is composed of repeating glucose units linked by p-1,4-glycosidic bonds. The individual molecules are held together by hydrogen bonds, forming largely crystalline fibres. The hemicellulose, which is a low molecular weight heteropolysaccharide, coats and binds the cellulose microfibrils, preventing the cellulose from becoming too crystalline. Three predominant types of hemicelluloses are recognised, namely 1,3- and 1,4-p-D-galactans, 1,4-p-D-mannans and 1,4-p-D-xylans, which are named according to the sugar type that forms the polymer backbone. Pectic substances contain rhamnogalacturonan backbones in which 1,4-linked a-D-galacturonan chains are interrupted at intervals with a-L-rhamnopyranosyl residues carrying neutral side chains. Two groups of enzymes, cellulases and pectinases, are required for the microbial utilisation of crystalline cellulose and pectin. Cellulases are multicomponent complexes that are often composed of endoglucanases, exoglucanases and cellobiases. Cellobiose is the major end product of concerted endoglucanase and exoglucanase activity. Cellobiose is then hydrolysed to glucose by p-glucosidases. The enzymatic breakdown of pectic polymers occurs by the deesterifying action of the saponifying enzymes, pectinesterase, releasing the methyl groups of the pectin molecule, and by hydrolase or lyase action of the depolymerases (pectin lyase, pectate lyase and polygalacturonase), splitting the a- 1.4-glycosidic linkages in the polygalacturonate chain. The yeast Saccharomyces cerevisiae has been used extensively in the alcoholic beverage industry for fermentations of wine, beer and other alcoholic beverages for many years. However, it is unable to produce extracellular depolymerising enzymes that can efficiently degrade polysaccharides, which are the main cause of clarification and filtration problems. Enzyme preparations have been used in the alcoholic beverage industries to degrade haze-forming polysaccharides, thereby improving the filterability and quality of products such as beer and wine. An alternative would be to develop S. cerevisiae strains that produce extracellular polysaccharidases, enabling the yeast to degrade polysaccharides without the addition of commercial enzyme preparations. These strains can also be very useful in improving the quality of wine, as well as cutting the costs of the winemaking process. The objective of this study was to investigate the effects of two transformed S. cerevisiae strains on different wine grape varieties. The following genes have been cloned and characterised previously: the Aspergillus niger endo-p-xylanase gene (xynC), the Butyrivibrio fibrisolvens endo-|3- 1.4-glucanase gene (endl), the Erwinia chrysanthemi pectate lyase gene (pelE) and the Erwinia carotovora polygalacturonase gene (p e h l). The yeast alcohol dehydrogenase I gene promoter (ADH1p), the alcohol dehydrogenase II gene terminator (ADH2j), the tryptophan synthase gene terminator (TRP5r) and the yeast mating-type pheromone a-factor secretion signal sequence (MFcrfs) were used to compile the following gene constructs: ADH1 p-MFa1 s-end1-TRP5r (designated END1), A DH1 p-xyn C-A DH2T (designated XYN4), ADH1 p-MFa1 s-peh1 -TRP5t (designated PEH1) and ADH1 p-MFa1 s-pelE-TRP5r (designated PELE). Two yeast integrating plasmids were constructed, one containing the END1 and XYN4 gene cassettes and the other containing the PEH1-PELE cassette. These two plasmids were then integrated into the URA3 locus of two separate industrial wine yeast strains of S. cerevisiae. To facilitate selection of the industrial yeast transformants in the absence of auxotrophic markers, the integrating plasmid containing the END1 and XYN4 gene cassettes was issued with the dominant selectable Geneticin G418-resistance {G f) marker. The integrating plasmid harbouring the PEH1-PELE gene cassette was issued with the dominant selectable sulphumetronmethyl resistance (SMR1) marker. The introduction of these plasmids into commercial wine yeast strains directed the synthesis of END1, XYN4, PELE and PEFI1 transcripts and the production of extracellular biologically active endo-P-1,4- glucanase, endo-(3-xylanase, pectate lyase and polygalacturonase. These recombinant yeasts were capable of extracting more colour from grape skins of certain varieties, as well as leading to more freeflow wine as a result of the more effective degradation of glucans, xylans and pectins in the skins. They also led to decreased turbidity in the wine, making it more filterable. Future work will entail further investigation of the effects of these recombinant yeasts on different white and red wine grape varieties. Another objective of this study was to screen non-Saccharomyces wine yeasts for the production of extracellular hydrolytic enzymes. The reason for this part of the thesis was to determine the types of extracellular hydrolytic enzymes that are produced and to determine which genera produce which kinds of extracellular enzymes. A total of 237 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces, were screened for the production of extracellular pectinases, proteases, (3-glucanases, lichenases, p-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts were all isolated from grapes and clarified grape juice to ensure that they were yeasts found in must during the initial stages of fermentation. This information can be used to pave the way to pinpoint the specific effects in wine of these enzymes produced by the so-called wild yeasts associated with grape must. This information can also be used to transform Saccharomyces wine yeasts with some of the genes from these non-Saccharomyces yeasts for the production of extracellular hydrolytic enzymes. However, future research will have to be done to determine the extent of the activity of these enzymes in wine fermentations and to obtain better knowledge of the physiological and metabolical features of non-Saccharomyces yeasts.

AFRIKAANSE OPSOMMING: Sellulose is verreweg die volopste koolhidraat in plantbiomassa. Hierdie biopolimere is dus ‘n baie belangrike hernubare bron van voedsel, brandstof en chemikaliee. Sellulose is in 'n matriks van hemisellulose, lignien en pektien gebed en is uit herhaalde glukose eenhede, wat deur middel van (3-1,4-glukosidiese bindings geheg is, saamgestel. Die individuele molekules word deur waterstofbindings aan mekaar geheg, wat aanleiding gee tot die vorming van kristallyne vesels. Die hemisellulose, wat 'n lae molekulere gewig heteropolisakkaried is, bedek en bind die sellulose vesels en verhoed daarmee die vorming van vesels wat te kristallyn is. Drie predominante tipes hemisellulose word herken en sluit 1,3- en 1,4-p-D-galaktane, 1,4-p-D-mannane en 1,4-p-D-xylane in, wat vernoem word volgens die suikereenhede wat die polimeerruggraat vorm. Pektiene bestaan uit 'n rhamnogalakturonaanruggraat waarin 1,4-gekoppelde a-D-galakturonaankettings periodiek met a-L-rhamnopiranosiel residue, bevattende neutrale sykettings, onderbreek word. Twee groepe ensieme, nl. pektinase en sellulase, word deur mikrobes vir die benutting van kristallyne pektinase en sellulase vereis. Sellulase is multikomponent komplekse wat dikwels uit endoglukanase, ekso-glukanase en sellobiase saamgestel is. Sellobiose is die hoof eindproduk van die saamgestelde aktiwiteit tussen endoglukanase en ekso-glukanase en word verder gehidroliseer tot glukose deur |3-glukosidases. Die ensimatiese afbraak van pektien polimere vind deur die de-esterifiserings aksie van die versepings ensiem, pektienesterase, plaas. Dit lei tot die vrystelling van die metielgroepe van die pektienmolekuul. Deur die hidrolase of liase aksie van die depolimerase (pektien liase, pektaatliase en poligalakturonase), split die a-1,4-glukosidiese verbindings in die poligalakturonaatketting. Die gis Saccharomyces cerevisiae word al vir jare ekstensief in die alkoholbedryf vir die fermentasie van verskeie produkte, veral druiwe, gebruik. S. cerevisiae besit egter nie die vermoe om ekstrasellulere depolimiserende ensieme wat vir die effektiewe degradasie van polisakkariede verantwoordelik is, te produseer nie, wat die hoof oorsaak van die verhelderings- en filtreringsprobleme in onder andere wyn en bier is. Dit veroorsaak ook dat S. cerevisiae nie oor die vermoe beskik om waasvormende polisakkariede in wyn te degradeer nie. Tans word ensiempreparate in die alkoholiese bedryf vir die degradasie van die probleem polisakkariede gebruik. Sodoende word die filtreerbaarheid en kwaliteit van wyn en bier verbeter. ‘n Goeie alternatief is die ontwikkeling van S. cerevisiae-rasse wat oor die vermoe beskik om ekstrasellulere polisakkarase te produseer en dus polisakkariede self sonder die byvoeging van eksterne kommersiele ensiempreparate te degradeer. Hierdie rasse sal baie voordelig wees vir die verbetering van wynkwaliteit, sowel as vir die vermindering van die kostes verbonde aan die wynmaakproses. Die objektief van hierdie studie is dus om die uitwerking van twee getransformeerde S. cerevisiae rasse, wat ekstrasellulere polisakkarases produseer, op verskillende wyndruifvarieteite na te vors. Die volgende gene is reeds voorheen gekloneer en gekarakteriseer: die endo-pxylanase- geen (xynC) van Aspergillus niger, die endo-p-1,4-glukanase-geen (endl) van Butyrivibrio fibrisolvens, die pektaatliase-geen (pe/E) van Erwinia chrysanthemi en die poligalakturonase-geen (p e h l) van Erwinia carotovora. Die alkoholdehidrogenase-geenpromotor (ADH1P), die alkoholdehidrogenase IIgeentermineerder (ADH2T), die gistriptofaansintase geen se termineerder (TRP5t) en die sekresiesein van die gisferomoon a-faktor (MFa1s) is gebruik om die volgende geenkonstrukte saam te stel: ADH1 p-MFa1 s-end1 -TRP5t (toekend as END1), ADH1 p-xynC-ADH2T (bekend as XYN4), ADH1 p-MFa1 s-peh1-TRP5T fbekend as PEH1), and ADH1 p-MFa1 s-pelE-TRP5T (bekend as PELE). Twee gisintegrerings plasmiede is gekonstrueer, een wat die END1- en XYN4- geenkassette bevat en die ander wat die PEH1-PELE-kasset besit. Hierdie twee plasmiede is daarna in twee aparte industriele wyngisrasse van S. cerevisiae by die URA3 lokus geintegreer. Vir die seleksie van die industriele wyngistransformante in die afwesigheid van ouksotrofiese merkers, is die dominante selekteerbare Geneticin G418 weerstandbiedende (G f) merker in die END1- en XYA/4-geenkassetbevattende plasmied geintegreer. Die dominante selekteerbare sulfumetronmetielweerstandbiedende (SMR1) merker is in die integreringsplasmied, wat die PEH1- PELE-geenkasset bevat, geintegreer vir seleksie. Transformasie van hierdie plasmiede in kommersiele wyngisrasse het tot die direkte sintese van die END1-, XYN4-, PELE- en PEH1-transkripte aanleiding gegee, sowel as tot die produksie van die biologies aktiewe ekstrasellulere endo-P-1,4-glukanase, endo-P-xylanase, pektaatliase en poligalaturonase. Tydens die wynmaakproses het bogenoemde rekombinante giste aanleiding gegee tot verhoogde kleurekstraksie uit die druifdoppe van sekere varieteite, asook tot verhoogde vryvloei wyn. Dit is verkry deur die effektiewe degradasie van die glukane, xilane en pektiene in die doppe. Die rekombinante giste het ook verlaagde turbiditeit in die wyn tot gevolg gehad, wat die wyne makliker filtreerbaar maak. Hierdie werk was net die eerste stap. In die toekoms sal verdere navorsing gedoen moet word om die presiese effekte van hierdie rekombinante giste op verskillende rooi en wit druifvarieteite te bepaal. ‘n Ander fokus van hierdie tesis was om nie-Saccharomyces wyngiste vir die produksie van ekstrasellulere hidrolitiese ensieme te selekteer. Die rede hiervoor is om te bepaal watter tipes ekstrasellulere hidrolitiese ensieme geproduseer word, asook watter ensieme deur watter genera geproduseer word, ‘n Totaal van 237 gisisolate wat tot die generas Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora en Kluyveromyces behoort, is vir die produksie van ekstrasellulere pektinase, protease, p-glukanase, lichenase, p-glukosidase, sellulase, xilanase, amilase en sulfiet reduktase-aktiwiteit getoets. Hierdie giste is almal vanaf druiwe en druiwesap geVsoleer om te verseker dat dit wel giste is wat gedurende die beginfases van fermentasie in die mos teenwoordig is. Hierdie inligting kan nou verder gebruik word om die spesifieke effekte wat hierdie ensieme, wat deur die sogenaamde wilde giste geproduseer word, tydens die beginfases van fermentasies op die mos het, te bepaal. Hierdie inligting kan ook in die toekoms gebruik word om Saccharomyces-wyngiste met gene van die ri\e-Saccharomycesgiste te transformeer om ekstrasellulere hidrolitiese ensieme vir die degradasie van die problematiese polisakkariede in wyn te produseer. Daar sal egter in die toekoms baie navorsing gedoen moet word om die omvang van hierdie ensiemaktiwiteite in wynfermentasies te bepaal, asook om meer kennis te bekom oor die fisiologiese en metaboliese samestelling van nie-Saccfraromyces wyngiste.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/53688
This item appears in the following collections: