Biotech in Wine biotech Sugarcane biotech South Africa Coal biotech...

ISSN 1860-6768 · BJIOAM 3 (11) 1321–1456 (2008) · Vol. 3 · November 2008

ReprintWine biotechSugarcane biotechCoal biotechPerspectives

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Biotech in South Africa

© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1355

Biotechnol. J. 2008, 3, 1355–1367 DOI 10.1002/biot.200800145 www.biotechnology-journal.com

1 Introduction

The wine industry in South Africa dates back to thearrival of Dutch settlers at the Cape peninsula in1652 (see [1] for a useful historical introduction).Jan van Riebeeck, first Commander of the Cape,was charged by the Dutch East India Company to

establish a refreshment station for Dutch shipstravelling to and from their colonies in East Asiausing the Cape sea route. He imported grapevinecuttings from Europe and began a small vineyard inthe company gardens, now situated in the city cen-tre of Cape Town.The first wines produced were re-ported to be of dubious quality, but with the arrivalof French Huguenots escaping persecution inFrance, important viticultural and winemakingskills were brought to the Cape. Dutch FreeBurghers and French Huguenots established vine-yards throughout the Cape peninsula with impor-tant settlements at Constantia, Stellenbosch andFranschoek.The beginning of the Nineteenth Cen-tury saw the occupation of the Cape by the British,which proved to be beneficial to the fledgling wineindustry. Wine from the Cape Colony was grantedpreferential access to the English import marketwhile wine from Napoleonic France was subject toheavy import duties. This period of growth was

Research Highlight

Wine biotechnology in South Africa: Towards a systemsapproach to wine science

John P. Moore, Benoit Divol, Philip R. Young, Hélène H. Nieuwoudt, Viresh Ramburan, Maret du Toit, Florian F. Bauer and Melané A. Vivier

Institute for Wine Biotechnology, Faculty of AgriSciences, Stellenbosch University, Matieland, South Africa

The wine industry in South Africa is over three centuries old and over the last decade has re-emerged as a significant competitor in world wine markets. The Institute for Wine Biotechnology(IWBT) was established in partnership with the Department of Viticulture and Oenology at Stel-lenbosch University to foster basic fundamental research in the wine sciences leading to applica-tions in the broader wine and grapevine industries. This review focuses on the different researchprogrammes of the Institute (grapevine, yeast and bacteria biotechnology programmes, andchemical-analytical research), commercialisation activities (SunBio) and new initiatives to inte-grate the various research disciplines. An important focus of future research is the Wine ScienceResearch Niche Area programme, which connects the different research thrusts of the IWBT andof several research partners in viticulture, oenology, food science and chemistry. This ‘FunctionalWine-omics’ programme uses a systems biology approach to wine-related organisms. The datagenerated within the programme will be integrated with other data sets from viticulture, oenolo-gy, analytical chemistry and the sensory sciences through chemometrics and other statistical tools.The aim of the programme is to model aspects of the wine making process, from the vineyard tothe finished product.

Keywords: Grapevine biotechnology · Systems biology · Wine biotechnology · Wine-omics · Wine science

Correspondence: Dr. John P. Moore, Institute for Wine Biotechnology,Faculty of AgriSciences, Stellenbosch University, Private Bag X1,Matieland 7602, South AfricaE-mail: [email protected]: +27-21-808-3771

Abbreviations: DVO, Department of Viticulture and Oenology; endoPG, en-dopolygalacturonase; FTIR, Fourier transform infrared; FTMIR, Fouriertransform mid-infrared; GC-FID, GC-coupled to flame ionisation detection;IWBT, Institute for Wine Biotechnology; KWV, Ko-operatiewe WijnbouwersVereninging van Zuid-Afrika; LAB, lactic acid bacteria; MLF, malolactic fer-mentation; NCED, 9-cis epoxy di-oxygenase; PGIP, polygalacturonase-in-hibiting protein; ROS, reactive oxygen species

Received 3 July 2008Revised 21 August 2008Accepted 29 September 2008

BiotechnologyJournal Biotechnol. J. 2008, 3, 1355–1367

1356 © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

short-lived as, towards the middle of the Nine-teenth Century, the protective tariffs granted byBritain were withdrawn, which coupled with polit-ical and economic turbulence, as well as the world-wide phylloxera epidemic, resulted in collapse ofthe Cape wine industry. The recovery of the wineindustry, however, was equally rapid, to the extentthat the lack of a suitable export market, previous-ly Britain, resulted in an overproduction that was ofdire concern to farmers and consumers alike. Toremedy this situation, co-operatives were formedto control price fluctuations and wine surpluses,the KWV (‘Ko-operatiewe Wijnbouwers Verening-ing van Zuid-Afrika’ or ‘Co-operative WinemakersAssociation of South Africa’) was formed in 1918with the task of regulating grape and wine salesthrough price control measures. During this period(early Twentieth Century) of market uncertainty,the first and still only university departments ded-icated to viticultural and oenological research inSouth Africa were established at the recentlyformed Stellenbosch University, previously VictoriaCollege, in the heart of the Cape winelands. It wasat the university experimental farm at Welgevallenin 1925 that the viticulturist, Professor Izak Perold,crossed Pinot noir and Hermitage (Cinsault) to cre-ate South Africa’s unique home-grown cultivar,Pinotage.The cultivar was painstakingly saved andpropagated from seedlings and was debuted anumber of years later on the South African winemarket. Currently, Pinotage remains South Africa’sflagship red wine cultivar with Cabernet Sauvi-gnon and Shiraz also being widely planted. To bol-ster basic research in South Africa, the Oenologicaland Viticultural research institute was establishedin 1955 at Nietvoorbij outside Stellenbosch.This in-stitute, currently part of the Agricultural ResearchCouncil of South Africa, concerns itself with basicviticultural and oenological research from a practi-cal ‘industry-based’ perspective. Similarly, the De-partments of Viticulture and Oenology at Stellen-bosch University for much of their history have fo-cused exclusively on improving basic viticulturaland oenological practices with a view to solving in-dustry relevant problems. With the re-entry ofSouth Africa into the international wine market in1994, the protective measures of the KWV were nolonger desirable. The dismantling of the price andsurplus control measures of the KWV, resulted inSouth African winemakers being subject to directcompetition with other major wine producingcountries for lucrative foreign markets.

The tremendous growth in wine sales and pro-duction post 1994 coupled with the pressures ofcompeting internationally has necessitated that theSouth African wine industry adapt to the changing

international environment. This required not onlyupdating viticultural and oenological practices andcomplying with international regulations, but alsothe development of high-level skills to further in-novation in grapevine and wine science. Biotech-nology, because of its cross-cutting nature as a re-search tool, its tremendous innovation potentialand its ability to attract students from differentfields to wine-related research was seen as one ofthe most important drivers to provide momentumto the transformation of the South African wine in-dustry. For this reason, the wine industry, support-ed by funding from the national government andthe University of Stellenbosch established the In-stitute for Wine Biotechnology (IWBT) in 1995 withProfessor Isak S. Pretorius of the Department ofMicrobiology as founding director. The Institute isaffiliated with the Department of Viticulture andOenology (DVO) with a vision and mission to be-come a nationally and internationally competitivecentre of excellence in wine and grapevinebiotechnology that, by means of visionary trainingand innovative research, provides the SouthAfrican grapevine and wine industry with well-trained human resources, cutting-edge technology,expert knowledge; and environmentally friendlyproducts and practices (see Fig. 1). The IWBT is acentre of postgraduate research in biotechnologyin South Africa providing training to honours, mas-ter and doctoral degree students with basic sciencebackgrounds (such as biochemistry, microbiology,chemistry, botany, etc.), as well as those comingfrom the applied agricultural sciences (such as viti-culture and oenology). The research portfolio cov-ers fundamental investigations into the cellularand molecular biology of wine-related organisms(grapevine, yeast and wine bacteria) and the appli-cation of biotechnological tools to improve theseorganisms. These tools range from traditionalmethodologies such as breeding and selection tothe use of genetic modification. The industry cur-

Integration of research disciplinesIntegration of research disciplines

Viticulture Oenology

Biotechnology

Long-term, strategic “blue sky” research

Short-term, grassroots-level research

Figure 1. A schematic representing how the biotechnology programme ofthe IWBT integrates into the DVO to position the South African wine in-dustry at the cutting edge of scientific innovation.


rently maintains that the IWBT and its researchportfolio creates the opportunity to competitivelyposition the South African industry and to reap thedirect benefits of GM products should world mar-ket perceptions change.

This review summarises the past and currentresearch thrusts of the IWBT, important publica-tions, new initiatives towards integrated research,as well as an initiative to facilitate the commercial-isation of IWBT research through the developmentof a biotech spin-off company.

2 Grapevine molecular physiology andbiotechnology

The grapevine biotechnology programme of theIWBT was started 10 years ago with the aim of sup-plementing and strengthening the few ongoing ef-forts in this field in South Africa [2]. Developmentof suitable cultivar-specific tissue cultures, trans-formation and regeneration systems and the basicmolecular biological tools to manipulate thegrapevine became the first focus of the programme[2, 3].

Grapevine is a woody perennial and has beenconsidered “difficult to work with” by geneticists,molecular biologists and biotechnologists. Woodyperennials, such as Vitis spp. have successive an-nual cycles of vegetative and reproductive growthwith intermittent dormant (winter) periods.Grapevine also exhibits extensive youth phasesthat significantly prolong generation times.The Vi-tis genome is of moderate size, but is consideredextremely heterogeneous and quite complex.These inherent characteristics of Vitis spp. remaincomplicating factors when studying grapevine, al-though the recent release of the grapevine genomesequence has provided new avenues for researchprogress [3, 4]. The genome sequence release hasprovided the impetus to develop Vitis as the first‘model’ woody perennial fruit crop [3, 4]. New sci-entific possibilities through the development ofmolecular tools (e.g. BAC libraries, molecularmarkers, genetic and physical maps) and theprospect of employing systems biology approacheshave arisen [3, 4].

Targeted gene disruption to create knockoutmutants and high throughput transformation pro-cedures are both still lacking in grapevine, makingsystematic analyses of signal transduction path-ways and epigenetic analyses difficult. Gene si-lencing mechanisms are also still being optimisedfor grapevine and virus-induced gene silencing,specifically linked to transient transformationtechnologies are one of the aspects targeted for de-

velopment in the international grapevine researchcommunity [2, 3].

Unlike model plant transformations, grapevinetransformation and regeneration is not yet routine.However, a number of cultivars and rootstocks havenow been successfully transformed and the firstfield trials of genetically manipulated grapevinehave been conducted. We have also been success-ful in developing transformation and regenerationplatforms for V. vinifera and are amongst the fewlabs worldwide able to routinely produce trans-genic grapevine tissue and plants [5].

As in all scientific fields, the viticultural sciencesneed hypothesis-driven research, facilitated by ex-perimental systems that yield repeatable resultswith a clear separation between cause and effect.Vineyard complexity, however, is an impediment tothis very basic requirement of scientific inquiry,leading to datasets that are difficult to interpret andextract statistically significant information. Fur-thermore, the results obtained are usually limitedto the specific vineyard and vintage under study.What is needed is a highly characterised vineyardwhere as many contributing factors are identifiedand measured.The concept of a ‘model’ vineyard tosupport hypothesis-driven research in viticulturalscience is one of the core drivers of a recently es-tablished integrated research programme, theWine Science Research Niche Area (RNA) that isdiscussed later in the review [6].

The main thrusts of the IWBT’s grapevinebiotechnology programme centres around plantstress (biotic and abiotic) and the consequent ef-fects on growth and fruit quality (see Fig. 2 for ex-amples of genes isolated and studied in thegrapevine programme). The fundamental ques-tion(s) we are trying to answer relates to the mo-lecular regulation of stress in grapevine. Bioticstress research focuses largely on fungal diseases,such as grey rot (Botrytis cinerea) and the mildews(powdery and downey) [7], whereas virus researchis conducted in a collaborating laboratory in theDepartment of Genetics at Stellenbosch Universi-ty. Fungal pathogens and insect pests continue tobe one of the most limiting factors in grapevine cul-tivation [7]. The production of fungal disease-re-sistant plants using transgenic technology is an at-tractive alternative to chemical treatments andshould encourage environmentally friendly prac-tises in the vineyard. To achieve this the IWBT hasfocused research efforts on chitinases [8, 9], poly-galacturonase-inhibiting proteins (PGIPs) [10, 11]and antifungal peptides [12], which are known toconfer disease resistance to host plants producingthese proteins. PGIPs are known to confer reducedsusceptibility to their respective hosts and the

Biotechnol. J. 2008, 3, 1355–1367 www.biotechnology-journal.com



PGIP-encoding gene from grapevine (Vvpgip1) isno exception [10, 11]. When expressed in a modelplant system such as tobacco, the protein confers aclear advantage to plants challenged with B.cinerea, leading to significant improvements in dis-ease resistance [10, 11]. The mechanisms by whichPGIP confers reduced disease susceptibilityagainst B. cinerea and fungal pathogens are not ful-ly understood. One mechanism thought to be in-volved is through the direct inhibition of cell wallmaceration enzymes secreted by fungal pathogens[10, 11]. The disease protection can be clearly ob-served by comparing tobacco plants overexpress-ing a potent endopolygalacturonase (endoPG)from Botrytis, leading to severe tissue macerationand plant structural collapse, whereas plants co-expressing both the endoPG and the PGIP displaya far less severe phenotype [10, 11]. Experimentalevidence also suggests that PGIPs could have func-tions totally unrelated to their activity, but still im-portant for plant defence [11]. Tobacco lines har-bouring the grapevine PGIP have been shown tohave an up-regulated disease resistance as well assignificantly more lignin than untransformed con-trols (unpublished observations).This wall-associ-ated phenotype is also reflected on a transcription-al level and genes involved in cell wall biosynthe-sis and structural organisation in transgenictobacco over-expressing PGIP (unpublished ob-servations). These results have spurred further re-search into identifying the nature of the direct andindirect mechanisms that may be responsible for

these disease resistance phenotypes. NumerousPGIP genes have been cloned and sequenced fromV. vinifera as well as wild Vitis species and are cur-rently being investigated for improved resistanceactivity.

Similarly, the first antifungal peptides fromgrapevine (VvAMP1) have been cloned. Purifiedpeptide has been tested against economically im-portant grapevine pathogens, showing significantactivity against several of them [12]. Antifungalpeptides are believed to mediate their activitythrough targeting the membrane structures of in-vading fungal organisms leading to pathogendeath. The exact mechanisms, in a similar vein tothe PGIP mode of action, appear to be the result ofdirect and indirect action on cellular processes.An-tifungal plant peptides and their encoding genesare abundant in most plant species and have a rec-ognized biotechnological potential in both themedical and agricultural biotechnology sectors.These peptides typically contribute to preformeddefence by developing protective barriers aroundgerminating seeds or between different tissue lay-ers within plant organs.

South Africa has a rich and unique floral biodi-versity and some native Brassicaceae spp. were tar-geted for the potential isolation of additional novelantifungal peptides. Fourteen new plant defensinsequences from four genera of the Brassicaceaefamily present in South Africa were isolated. Mem-bers of this group are well known for their strongantifungal activity, but other activities such as met-

Polygalacturonse-inhibiting proteins: Vvpgip1, 38 pgip genes from Vitaceae and Muscadinia.

Antifungal peptides: Vv-AMP1, MiAFP1-MiAFP3, HcAFP1-HcAFP6, LaAFP1-La-AFP4, LmAFP1-Lm-AFP2.

General defense genes: vst1, Ntcad, CST1-2, EXG1.

Antifungal/disease resistance (against bacterial, fungal and insect pests)

Carotenoid biosynthetic pathway: VvPSY , VvPDS, VvZDS, VvCiso, VvLECY , VvECH, VvLBCY,

VvBCH, VvZEP , VvVDE , VvNSY .Abscisic acid biosynthetic pathway: VvNCED , VvCCD.

Increased abiotic stress tolerance (drought, light) and/or fruit quality (flavour, aroma)

Mevalonate pathway: VvAACT, VvHMGS, VvHMGR , VvMK, Deoxyxylulose 5-diposphate pathway: VvDXS , VvDXR , VvispE, VvispF, VvispG, VvispH,

General: VvIPI, VvFPS, VvGPS, VvGGPS.

Increased isoprenoid levels/general metabolism (multiple metabolic functions)

GRAPEVINE BIOTECHNOLOGY

CAT2, YAT1, YAT2, CIT2, AGP2, ATF1, ATF2, BAT1, BAT2, GPD1, GPD2 , AAT10, AAT11.

Aroma compound metabolism (improved sensory qualities)

MSS10, MSS11, FLO1, FLO5, FLO8, FLO10, FLO11/MUC1, CLN1, CLN2, GPA2, ASH1,

STE7, STE11, STE12, TEC1, MSN1, NRG2, ROM1, ROM2, SOK2, KSS1, PH D1, RAS2,

RME1, SCH9.

Modified adhesion properties (flocculation, sedimentation and turbidity in wine)

STA1/DEX2/MAL5, EXG1/BGL1, BGL2, SSG1/SPR1, EXG2, PAD1/POF1, CTS1-2, PEP4,

ATF1/IAH2, PGU1/PGL1, STA2/DEX1, STA3/DEX3, SGA1, LKA1, LKA2, S FA1, SFG1, RSA1,

RSG1, XYS1, XYN2, XYNC, XYN5, BGLA, XYL1.

Extracellular enzymes to improve processing and wine quality

YEAST BIOTECHNOLOGY

Figure 2. Examples of genesisolated in the IWBT yeast andgrapevine biotechnology pro-grammes in the last 10 years.


al tolerance and inhibition of protein translationare also known. The unique genetic resources ob-tained in this study have entered our biotechnolo-gy programme where improved disease resistanceof grapevine is the main aim.

The utility of single genes encoding specificproteins, PGIP or antifungal peptides, with markedanti-fungal activity genetically engineered intograpevine is an extremely useful technology for thecontrol of vineyard pathogens [2, 7]. Further re-search aimed at understanding the dynamics ofplant-pathogen interactions and the role of pro-teins with antifungal activity will offer better in-sights into the potential of utilising this technologyin plant protection strategies.

Apart from the common biotic threats, viruses,fungi and insects, grapevine plants are susceptibleto abiotic stresses caused by environmentalchange. One common type of abiotic stress found inSouth Africa is drought. Southern Africa is facingprogressively worsening water shortages with theresult that crop productivity suffers, thus negative-ly influencing the regional economy. Grapevine isno exception where water stress can significantlyimpact factors such as canopy growth and bunchquality. Water stress also effects properties such asphotosynthetic efficiency and can promote photo-oxidative stress resulting in reactive oxygenspecies (ROS) production. To develop grapevineswith enhanced capabilities to grow under adverseconditions, in particular water stress, the IWBTgrapevine programme is utilising genetic resourcesdeveloped by studying the carotenoid biosyntheticpathway in grapevine. This pathway producesmetabolites or compounds involved in environ-mental stress responses (antioxidant molecules),quality parameters of grapes (aroma molecules)and the formation of the stress hormones (such asabscisic acid). The understanding and manipula-tion of carotenoid metabolism in grapevine willprovide insight into how grapevine deals with abi-otic stresses, but can also provide alternativestrategies to counteract, and/or produce novelplant material more resistant, to these stresses.Moreover, the work also aims to contribute towardsa fully characterized vineyard where the cause(s)and effects of environmental stresses can be sepa-rated and studied.The metabolic pathways that willbe studied are also intricately involved in qualityaspects and so some of the research objectives re-late to functional analysis of impact genes and theirencoded products to improve flavour and aromaproduction in grapevine tissues and berries.

Carotenoids play a central role in plant metab-olism in general, and specifically in photosynthesiswhere they perform a myriad of functions. In addi-

tion to being integral structural and accessorylight-harvesting components of the photosyntheticapparatus, their ability to quench ROS formed un-der adverse conditions, thereby protecting the pho-tosynthetic machinery against damage, is invalu-able. Carotenoids also serve as important precur-sors for apocarotenoids. These apocarotenoids ornorisoprenoids are involved in a wide range offunctions in plants and can be growth regulators,pigments, flavours and aromas. Abscisic acid is animportant apocaroteniod involved in growth regu-lation and stress responses. Norisoprenoids arefrequently aromatic and volatile in nature and con-tribute to the varietal character of a number of im-portant cultivars while having very low odour de-tection thresholds.

Work in the grapevine programme has lead tothe cloning and characterisation of most of thegenes encoding enzymes involved in the pathway[13–16]. The role of these enzymes have been as-sessed in further studies by overexpression of se-lected genes in tobacco and Arabidopsis [15]. Theresultant transgenic plants have been assessedgenotypically and phenotypically characterised toprovide us with insights into the functional role ofthese genes in plants. These analyses revealed anumber of parameters involved or affected duringdrought tolerance. Collectively, these results con-firm and advance our existing knowledge of therole of carotenoid biosynthesis in drought toler-ance and the specific mechanisms operating on awhole plant level.

A clear understanding of the respective genes,proteins and enzymes that contribute to thecarotenoid biosynthetic pathway is needed to suc-cessfully apply genetic manipulation strategies. Tosupport this research, a grape berry tissue culturesystem is being developed.The aim is to determinewhether such a system would be suitable as modelsystem to investigate berry ripening and otherberry-specific processes. In summary, the currentprojects focus on improving our understanding ofgenetic networks and mechanisms responsible forthe plant’s response to biotic and abiotic (environ-mental) stresses, as well as the aroma and flavourdevelopment in grape berries and the regulationthereof. Systems biology tools are increasingly im-plemented into the programme as they becomeavailable.

The datasets generated will also allow carefulevaluation of current manipulation strategies andsupport the development of improved approachesin the future.




3 Yeast molecular biology and biotechnology

The IWBT has a long history of research projectson improving yeast strains for specific applicationsusing molecular genetic and breeding technology[17]. Yeast projects are focused on establishing abetter fundamental understanding of yeast cellularand molecular biology, and to apply this knowledgeto generate new wine yeast strains.Topics of inves-tigation include gene regulation and signal trans-duction networks, the molecular nature of cell wall-related phenotypes, metabolic regulation and thesecretion of enzymes (see Fig. 2 for examples ofgenes isolated and studied in the yeast pro-gramme).

These fundamental projects feed into thebiotechnology program, which uses traditionalmethodologies of breeding, selection, directed evo-lution and molecular modification. Applied proj-ects in the past focused on engineering yeasts withspecific enzymes useful in the degradation of di-verse polysaccharides [18–21], studying the prop-erties of yeast adhesion phenotypes [22–28] thatare of interest during fermentation processes inyeast during winemaking [29], the genetics of car-bohydrate source utilisation (e.g. starch, cellulose)[30–33], the metabolic engineering of aroma pro-duction pathways [34, 35], carnitine production [36,37] and risk assessment related to the use of genet-ically modified wine yeast strains [38].

The IWBT has combined traditional technolo-gies with directed evolution to generate new strainsof industrial relevance. Directed evolution refers tothe application of specific selection pressures overmany generations of yeast growth usually in a con-tinuous fermentation chemostat system. In such asituation strains that evolve advantageous adapta-tions to the selection pressure out-compete lesswell adapted strains. Through this approach theIWBT has been able to generate yeast strains withspecific properties such as improved nitrogen effi-ciency or fructose utilisation. The success of theyeast breeding programme is evident in the num-ber of valuable strains produced by the IWBT forindustrial applications. An example being the suc-cessful VIN13 Saccharomyces cerevisiae strain mar-keted by Anchor Yeast and used in wine fermenta-tions both in South Africa and abroad.

Although traditional breeding methods provideuseful strains of industrial relevance, this particu-lar approach is limited in two major ways. Firstly,such traditional methods can only improve pre-ex-isting characteristics present in the yeast, not gen-erate new traits that may be desirable. Secondly, ex-perience has shown that even traits within yeastcan be improved up to a specific point while further

improvement is only possible through the integra-tion of additional genetic material.A further disad-vantage of these technologies is their inherent ran-domness, which makes outcomes unpredictable. Itis also difficult to breed strains for traits that do nothave an easily selectable character as is in the caseof strains with specific aroma production capabili-ties. To overcome these limitations, techniques ofmolecular biology and genetic engineering are em-ployed.

The specific aims of the current yeast strain de-velopment programme integrates systems biology-based approaches, and also continues to pursuestrategies based on traditional breeding or geneticmodification technology. The programme includesmany of the areas that have been identified as be-ing of commercial relevance for the yeast and wineindustries. The programme can be subdivided intofive major research themes, these being: (i) pro-ducing yeast with greater fermentation efficiency,(ii) improving wine processing and filtration, (iii)developing yeast that improve the aroma andflavour of wine, (iv) increasing the wholesomenessof wine, and (v) improving wine preservation. Eachof these programmes support the main thrust ofthe yeast biotechnology programme, which is togenerate and assess new yeast strains of potentialvalue to the South African wine industry.

A focus of the current program is the generationof yeast that would yield lower levels of ethanol.South Africa is a warm climate region with the re-sult that harvested grapes produce on averagehigher levels of sugar than cool climate wine re-gions. This results in wine with higher-than-aver-age alcohol levels due to the greater amount of fer-mentable sugar in the must. Export markets aremore favourable to moderate alcohol levels in wineand thus the ability to control alcohol production isnecessary.To this end the IWBT developed a strat-egy in which the enzyme glucose oxidase is engi-neered into yeast to convert the excess sugar (glu-cose) present during fermentation into glucono-lactone and so prevent excessive alcohol produc-tion [39]. In addition, during the fermentation ofmust, numerous partially soluble complex plantpolysaccharides are extracted into the wine medi-um. These polysaccharides are viscous and tend toblock filters as well as impede processing stepsduring winemaking.The general type of plant poly-saccharides present can be divided into cellulose,hemicellulose and pectin polymers.A genetic engi-neering solution to these problems consisted of en-gineering polysaccharide-degrading enzymes suchas polygalacturonases, xylanases and cellulasesinto yeast using molecular secretion systems[19–21, 40].Thus, yeast are able to ferment the must


normally to produce wine while simultaneously de-grading viscous polymers thus significantly en-hancing processing steps such as filtration. Yeastengineered to degrade polysaccharides to improvefiltration are also able to increase the ‘fruity’ bou-quet of the wine. Wine yeast strains produce manyof the aroma and flavour compounds that definethe individual character of specific wines [41].These compounds include in particular esters,higher alcohols and various sulphites, as well asacids, glycerol and terpenoids. Genetic and meta-bolic engineering strategies are able to modify themetabolic flux in a particular pathway in such away as to favour the production of desirable com-pounds while concomitantly reducing undesirableones. The power of a metabolic engineering ap-proach is evident when considering that the over-expression of single genes are able to significantlymodulate the levels of over a dozen important aro-ma compounds in yeast simultaneously [41–44].Similar data have been generated for many genes,allowing a better understanding of the interactionsbetween many metabolic pathways involved in aro-ma compound production [43, 44]. In addition toaroma compounds, yeast has the potential to pro-duce compounds of human medical importance.Grape-derived compounds, such as the phenoliccompound resveratrol, is linked to the health ben-efits of moderate wine consumption [45]. Resvera-trol is positively correlated epidemiologically withreduced cardiovascular disease and so the IWBTwas interested in increasing the amounts presentin wine using a designer yeast strategy [45]. Twogenes from the metabolic pathway needed to pro-duce resveratrol (a coenzyme A ligase-encodinggene, 4CL216, from hybrid poplar and thegrapevine resveratrol synthase gene, vst1) werecloned into a laboratory strain of S. cerevisiae [45].The results obtained by analytical liquid chro-matography-coupled mass spectrometry demon-strated that the yeast transformants were able toproduce piceid, which is the glucose-bound form ofresveratrol [45]. These yeasts therefore have theability to produce resveratrol during fermentationin both red and white wines, thereby increasing thewholesomeness of the final product. Although mi-crobes, such as the resveratrol-producing yeaststrain, impart positive attributes to wine, a commoncause of wine spoilage is the growth of micro-or-ganisms such as bacteria that have the potential toproduce off-flavours [45]. A common practise toprevent such spoilage is the addition of sulphurdioxide as a preservative. While sulphur dioxide issatisfactory in this regard, and in addition also actsas an antioxidant, alternatives are desired and ac-tively encouraged because of the potentially nega-

tive influence of the compound on aroma andhealth, a significant percentage of the populationbeing hypersensitive to sulphur dioxide.The IWBThas generated yeast strains that control the growthof spoilage organisms by secreting peptides or en-zymes that specifically inhibit the growth of un-wanted organisms [46]. An example being the useof bacteriocin genes cloned into a laboratory strainof yeast [47]. Two bacteriocin-encoding genes, pe-diocin PA-1 (pedA) produced by Pediococcus acidi-lactici and leucocin B (lcaB) from Leuconostoccarnosum, were cloned into a multicopy episomalplasmid under the control of the alcohol dehydro-genase 1 promoter and terminator, and the yeastmating pheromone _-factor secretion signal [47].Transformed yeast strains were shown to produceactive forms of pediocin and leucocin [47]. Thesebactericidal yeast strains were not only able to con-duct fermentations in wine, but also act as a biolog-ical control agent by inhibiting the growth ofspoilage bacteria [47]. Although certain bacteriaare able to cause wine spoilage, some bacteria im-part beneficial properties to wine and are thusadded to wine formulations to encourage growth.

4 Lactic acid bacteria biotechnology

Lactic acid bacteria (LAB) have historically beenassociated with food and beverage fermentationsas they occur naturally in the starting materialsused [46]. Lactic acid bacteria also occur in mustand wine and perform the secondary fermentation,known as malolactic fermentation (MLF). Theprocess of MLF includes a reduction in acidity, re-sulting from the degradation of L-malic acid to L-lactic acid with the concomitant release of carbondioxide. LAB isolated from grapes and wines arefrom the genera Lactobacillus, Leuconostoc, Oeno-coccus and Pediococcus. Oenococcus oeni is usedcommercially in malolactic starter cultures, as theyare the LAB best adapted to wine conditions. Al-though MLF is primarily performed to reduce wineacidity, especially in cooler climate regions, it is alsoconsidered beneficial to the wine’s sensory qualitydue to flavour modification. Additionally MLF pro-vides microbial stability, since malic acid, which canserve as a carbon source to support the growth ofpotential spoilage LAB, is degraded [48]. In addi-tion, LAB produce antimicrobial agents that pro-tects the finished product by inhibiting spoilagebacterial growth [49]. These bacteria are able tocompete for nutrients during fermentation and canproduce antimicrobial compounds such as organicacids, ethanol, hydrogen peroxide and bacteriocins[46, 50]. A wide range of LAB have the ability to




produce bacteriocins and the fundamental knowl-edge gained on the biochemical and genetic char-acteristics of these molecules in the last decade hasincreased their potential application as biopreser-vatives in the food and beverage industries [46, 50].

Internationally, rapid progress has been madein the last 10 years in the development of tools forthe genetic modification of LAB. The major targetof the LAB strain development programme of theIWBT is to select for strains that are better adapt-ed as starter cultures for MLF and to better under-stand the physiology, diversity and performance ofstrains under winemaking conditions. Advanceshave been made in the development of transfor-mation systems involving the development of inte-gration and amplification vectors, selection criteriaand food-grade heterologous expression systems.The genome sequences of several LAB have be-come available contributing to the study of genesand operons in these important bacteria. Researchefforts at the IWBT are focused on (i) specific en-zymes that are involved in the production of winearoma compounds, (ii) bacteriocins that can beused as alternative to chemical preservatives, (iii)investigating the role of LAB in bitterness, (iv) pro-duction of biogenic amines, and (v) compoundscausing off-flavours in wine.The research findingswill be useful in the identification of target genesfor the future selection of improved starter culturesor the direct genetic improvement of LAB strainsused in MLF.

The first aim involves the screening of naturalwine LAB strains, as well as commercial starter cul-tures for enzymes important in the production ofwine aroma compounds. The enzymes focused oninclude _-glucosidases, proteases, esterases, glu-canases, citrate lyases and enzymes involved in theproduction of off-flavours such as volatile sulphurand phenols [51, 52].Thus it is important to know ifthis aroma enhancing potential is realised underMLF conditions and to what extent MLF influenceswine flavour composition [51]. Genetic screeningwas employed to assess the presence of bacteri-ocin-encoding genes. PCR revealed the presence ofthe plantaricin encoding genes plnA, plnEF, plnJ,plnK in Lb. plantarum strains. Four putative bacte-riocin-encoding genes in the genome of O. oeniwere identified and sequenced [49]. It is alsoknown that certain LAB have the potential to affectthe wholesomeness of wine by producing biogenicamines [53, 54]. Biogenic amine production isstrain related due to the possession of the specificbiogenic amine decarboxylase gene, which is influ-enced by winemaking parameters [55].Therefore, amajor thrust was to investigate to assess gene dis-tribution and homology amongst strains.

Another contribution made by rare strains ofLAB is the ability to cause bitterness in wine. Cer-tain LAB possess the ability to degrade glycerol,leading to the formation of acrolein, an intermedi-ate that is able to react with phenolic groups of an-thocyanins, forming a bitter complex [56]. The keyenzyme involved is glycerol dehydratase (gdh) thatfortunately is not widespread in nature. From 240natural LAB isolates only 26 contained the geneand they belonged to Lb. plantarum, Lb. pentosus,Lb. hilgardii, Lb. paracasei, Lb. brevis and a Pedio-coccus spp.To our knowledge, this is the first reportof the presence of the GD gene in Lb. plantarum,Lb. pentosus and Lb. paracasei [56]. The main aimsof the LAB research programme is thus orientedtowards developing tools to investigate suitableLAB strains for commercial use in the SouthAfrican wine industry.

5 Chemical-analytical research

Since 2000 the IWBT has systematically acquiredanalytical instrumentation and developed re-sources to put the institute in the position to main-tain the forefront cutting-edge analytical capacityneeded in the biotechnological environment. To-day, the IWBT has an analytical facility that pro-vides full support for metabolomic and wine ana-lytical approaches, based on HPLC, capillary elec-trophoresis (CE), GC coupled to flame ionisationdetection (GC-FID), GC-MS and a range of spec-troscopic instruments.The laboratory is also devel-oping several high-throughput analytical tools.

The volatile composition of South African wineswas determined with GC-FID and a combination ofGC-FID and Fourier transform infrared (FTIR)spectra have been modelled by chemometric tech-niques to discriminate between the major wine cul-tivars. HPLC is used for quantification of sugars,ethanol, organic acids as well as for phenolic com-pounds in wine.

Fourier transform infrared (FTIR) spectrome-ters designed specifically for applications in thegrape and wine industries are recent additions tothe arsenal required to handle the demands onmodern wine chemistry.The IWBT was the first re-search institution to venture into this technology byacquiring a GrapeScan FTIR spectrometer in 2002.Since then, the IWBT has played an important rolein introducing the technology and its potential ben-efits to the South African wine industry, in assistingwith the implementation thereof and in facilitatingtraining of users by both local and international ex-perts.This methodology is now used with great suc-cess for routine wine and grape analysis, and it also


plays a strong supporting role for research at theIWBT and has established itself as a research focusin its own right.The first non-routine application ofFTIR at the IWBT was to establish a calibration forthe analysis of glycerol in wine [57, 58]. This ana-lytical tool was used to conduct an industry-widesurvey of the glycerol levels in premium SouthAfrican wines and to test the hypothesis that theglycerol concentration in this category wine is pos-itively linked to quality [57, 58]. FTIR spectroscopyis also a valuable tool in the IWBT yeast biotech-nology programme by speeding up the screening offermentation profiles of desirable variants within apopulation of wine yeast strains and to evaluatewine yeasts that have been genetically modified.FTIR spectroscopy established for quantitativeanalysis in wine is also now optimised for monitor-ing grape quality during ripening, yeast and mustduring alcoholic fermentation and bacteria causingMLF. Rapid detection of spoilage and the identifi-cation of wine-associated bacteria using FTIR-coupled attenuated total reflectance (ATR) is con-ducted with the long-term aim of establishingspectral libraries for future research projects.Fourier transform mid-infrared (FTMIR) spec-troscopy has also been used to establish fermenta-tion profiles of yeast strains developed in a classi-cal breeding programme for the enhanced produc-tion of glycerol [59]. FTMIR spectroscopy is firmlyestablished in the IWBT as an analytical tool forevaluation of the volatile fermentation profiles ofyeasts during breeding and fermentation. FTMIRspectroscopy has also been optimised for monitor-ing quality control parameters (sugar content, pHand titratable acidity) during grape ripening [60]and the current focus is on bioprocess monitoringincluding both alcoholic fermentation and MLF.The IWBT has recently acquired an near infrared(NIR) spectrometer, which has expanded the ma-trix that can be analysed to include not only liquids,but also semi-solids and solids. These latter appli-cations currently include the development of quan-titative calibrations on anthocyanin content ingrape-skin homogenates and quality parameters ofwhole grape berries (such as browning potential).Future research applications include cell wall bio-spectroscopy focused on grape berry ripeningprocesses, plant-pathogen interactions and yeastmannoprotein production.

To investigate and correlate the large amount ofdata generated using these spectroscopic ap-proaches with established chemical referencemethods requires advanced statistical support.Theextraction and interpretation of the relevant ‘un-derlying’ information in analytical datasets re-quires extensive application of chemometric tech-

niques. Modern instrumentation are frequently es-tablished with customized software packages thatincludes multivariate quantification, classificationand cluster analysis tools [61, 62]. The moderntrend to apply chemometric techniques to analyti-cal and instrumental signals has opened up thepossibility in biotechnology to establish algorithmsthat can be used for investigating trends, monitor-ing bioprocesses and for the prediction of futureoutcomes related to biological processes as well aschemical and sensory profiles. The IWBT is at theforefront of these initiatives with active interna-tional collaborators including industrial and aca-demic research partners to support the develop-ment of spectroscopic and chemometric tools in theSouth African wine environment. The IWBT hasalso been instrumental in establishing the SouthAfrican Chemometrics Society (SACS) with the as-sistance of local academics and internationalchemometric experts.Thus the IWBT plans to con-tinue to be at the forefront of initiatives to integratewine science research in South Africa into a coher-ent framework through the sustained developmentof the necessary instrumentation, statistical soft-ware tools and expert supported training of indus-try and academic stakeholders.

6 SunBio

SunBio is an exciting new venture that presents anopportunity to realise the commercial potential ofthe IWBTs research activities by establishing aspin-off company from Stellenbosch University.SunBio aims to establish a sustainable product de-velopment process that will combine the researchoutput and intellectual property generated by theIWBT with sound commercialisation practices.Thecompany will focus on the areas of genetic en-hancement technologies, conventional develop-ment of unique yeast and bacterial strains, and de-velopment of niche service offerings (chemical andmicrobiological support) to the wine industry. Sun-Bio was initiated by funding obtained from CapeBiotech Trust, a Biotechnology Innovation Centre(BIC) established by the South African Departmentof Science and Technology.

SunBio research projects are designed to gener-ate a large number of hybrid and/or recombinantwine yeast strains. For some projects this will in-volve the development of gene expression cas-settes and yeast strains that are able to enrichwines with antioxidants, and nutritional supple-ments geared toward the health and wellness mar-ket segment. SunBio, also aims to make the fer-mentation process more efficient through the pro-




duction of yeast strains with enhanced levels of keyfermentation enzymes, and reducing the relianceon sulphur dioxide during the fermentationprocess through the use of bacteriocins.

These research and technology developmentswill be an important strategic advantage for theSouth African wine industry in the global market.SunBio seeks to actively commercialise the noveltechnologies at the IWBT, thus contributing to-wards the global competitiveness of the SouthAfrican wine industry.

7 Functional wine-omics and futureperspectives

Biotechnological innovation has defined the activ-ities and goals of the IWBT since its inception andwill continue to be our core focus.The competitive-ness of the global wine industry, the increasing fo-cus on and adoption of new technologies in viticul-ture and oenology, the ability to virtually interactand share knowledge in the research community, aswell as strategic opportunities and technologicaladvancements in general necessitate an integratedapproach to wine and grapevine research. Biotech-nology will only be useful when integrated with thedisciplines of viticulture and oenology, and whensupported by the fundamental biological sciences,including biochemistry, chemistry, genetics, micro-biology and botany. Furthermore, technological ad-vances create exciting opportunities to incorporatenew methods and approaches in the wine sciences.Very basic research questions remain unansweredand hypothesis-driven research remains difficultin various fields of wine sciences due to the inher-ently high heterogeneity in the test systems.

How do we then move forward towards a scien-tific qualification of vine and wine productionwhile optimally employing new technologies, cre-ating exciting and novel opportunities for the mod-ern Wine Sciences? The newly approved Wine Sci-ence Research Niche Area (RNA) aims to makeprogress in this regard and proposes to integrateemerging technologies into the study of wine andwine organisms.As a methodological core the RNAhas been built around “Metabolomics and Metricsof Wine and Wine Organisms” (see Fig. 3 for a dia-grammatic summary of the Wine Science ResearchNiche Area).

This theme represents a multidisciplinary, inte-grated and, in its scope, internationally unique ef-fort that combines the power of new cutting-edgescientific tools of global molecular analysis (ge-nomics, transcriptomics, proteomics and meta-bolomics) with the existing physiological, biochem-

ical, genetic and molecular research at the IWBT. Inthe biological sciences, many approaches today arebased on global analysis tools, also referred to asOmics (genomics, proteomics, metabolomics), andon the holistic integration of data from differentdisciplines to further the understanding of thewhole (systems biology). The techniques, tools andthought patterns employed by those approachesare clearly of major relevance to wine science, andneed to be implemented on a large scale.

The specific research problems to be addressedare all directly related to issues that have beenidentified by the South African wine industry as themost relevant problems threatening the future via-bility of the industry. More particularly, the propos-al covers those aspects where biotechnological ap-proaches can provide a significant competitiveedge to the South African wine industry. These as-pects are (i) environmentally friendly productionpractices (disease-resistant grapevines), (ii) re-duced production costs (yeast with improved fer-mentation efficiency and improved resistance tonutrient stress), (iii) the production of wine of con-sistently high quality (better colour, flavour andaroma), and (iv) providing increased health bene-fits through metabolic engineering of yeast andother wine-associated microorganisms. Further-more, to contribute to the long-term economic via-bility of these approaches, potential risks that maybe associated with the release of GM organismshave to also be assessed.

Traditional “Wine Science”Chemistry

Core sciences

Applications

Wine Science Research Niche Area

AnalyticalChemistry

Methoddevelopment

ViticultureOenology

“Omics”

Biology of wineorganisms

MetabolomicsMetrics

Improved organisms Improved

methods and practices

Sensorial and chemical profiles

Biology

Wine Science Research Niche AreaWine Science Research Niche Area

Figure 3. A diagram representing the ‘Wine Science Research Niche Area’programme coordinated between the IWBT, DVO, Food Science andChemistry at Stellenbosch University. The model reveals how core disci-plines of biology, chemistry, viticulture and oenology are connected via(chemo)metric and metabolomic techniques to applied products andmeasurable outcomes (e.g. improved sensory profiles and fermentationprocesses).


A holistic approach towards wine scienceshould attempt to integrate all scientific disciplinesthat are relevant to the understanding of wine-as-sociated organisms. The two traditional wine sci-ences, viticulture and oenology, need to be com-bined with new approaches developed in thechemical, biological and other sciences, in particu-lar, analytical chemistry and biotechnology. On ascientific level, combined approaches promise todeliver a significantly improved understanding ofall the biological processes that determine indus-trially and commercially relevant parameters, costefficiency and product quality. Besides the scientif-ic and commercial benefits, South African scienceand the wine industry stand to gain in image andinternational appreciation as being innovative andforward looking. The IWBT with its integrated re-search programme entitled “Functional Wine-omics” will continue to develop the tools and ex-pertise required to be at the frontier of researchinto the systems biology of wine.

The authors have declared no conflict of interest.

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MALDI MS – The leading experts will guide you!

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FRANZ HILLENKAMP, University of Münster, Germany, and JASNA PETER-KATALINIC, University of Münster, Germany (eds.)

MALDI MS

A Practical Guide to Instrumentation, Methods and Applications

Written by one of the leading experts in the field, this first comprehensive book on MALDI-MS in life sciences provides an in-depth description of many different aspects and applications of this revolutionary technique.

The 2002 Nobel Prize was awarded for the development of methods for identification and structure analyses of biological macromolecules. MALDI is one of the two mass spectrometric methods besides Electrospray which is universally used for this purpose.

This unique book gives practical and educational asset for individuals, academic institutions and companies in the field of bioanalytics.

2007. XVI, 362 pages, 111 figures 26 in color, 7 tables. Hardcover. ISBN: 978-3-527-31440-9 € 99.- /£ 75.- /US$ 135.-

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