Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Humanity is currently dependant on fossil fuels as an energy source. Increasing economic development and industrialization is, however, raising the demand for this unsustainable energy source. This increased pressure on dwindling reserves and growing concern over detrimental environmental effects associated with the use of these fuels have sparked great interest in the development of alternative sources. Bioethanol has surfaced as a good alternative to fossil fuels, as it can be produced from cheap, abundant, renewable, non-food sources. Bioethanol is also carbonneutral, i.e. utilisation thereof leaves the net level of carbon dioxide in the atmosphere unperturbed. Lignocellulose, more specifically its cellulose fraction, has been identified as a possible feedstock for the production of bioethanol. The use of lignocellulose as feedstock will allow for a more sustainable supply and much needed energy security. Lignocellulosic feedstocks can be divided into two main categories, i.e. wastes from processes other than fuel production and crops grown specifically for fuel production. Cereal crops such as triticale have been identified as good industrial crops for the production of energy. Triticale’s higher biomass yield, moderate water and nutrient requirements, steadily increasing area of cultivation and main use as an animal feed and not a human food source, makes it attractive as feedstock for the production of bioethanol. The combined activity of endoglucanases, exoglucanases and β-glucosidases is needed to hydrolyse crystalline cellulose to fermentable sugars. The high cost of these enzymes is, however, the most significant barrier to the economical production of bioethanol from cellulosic biomass. A promising strategy for a reduction in costs is the production of these cellulolytic enzymes, hydrolysis of biomass and fermentation of the resulting sugars to bioethanol in a single process step via a cellulolytic microorganism. The development of such a consolidated bioprocessing (CBP) organism can be achieved by the introduction of cellulolytic activity into a noncellulolytic microorganism that is able to ferment glucose to ethanol. Saccharomyces cerevisiae is a good host candidate for CBP as this yeast’s high tolerance towards ethanol and its use in industrial applications has been established. The enzymatic activities of endoglucanases and exoglucanases are, however, inhibited by the build-up of cellobiose during the hydrolysis of cellulose. This effect may be alleviated with the introduction of a better functioning β-glucosidase into the system. β-Glucosidases hydrolyse cellobiose to glucose, alleviating the inhibition on the enzymatic activities of endoglucanases and exoglucanases. Despite advances in enzyme production systems and engineering enzymes currently in use for higher stability and activity, there is still a demand to expand the current collection of enzymes. Bioprospecting for novel cellulolytic enzymes focuses on specific environment, with high turnover rates of cellulosic material or extreme conditions, such as the composting process. These enzymes are becoming more attractive compared to their mesophillic counterparts due to their potential industrial applications and the fact that they represent the lower natural limits of protein stability. / AFRIKAANSE OPSOMMING: Die mensdom is hoofsaaklik van fossielbrandstowwe as 'n energiebron afhanklik. Toenemende ekonomiese ontwikkeling en industrialisasie verhoog egter die aanvraag na hierdie onvolhoubare energiebron. Druk op kwynende reserwes en groeiende kommer oor die nadelige gevolge vir die omgewing wat met die gebruik van hierdie brandstowwe gepaard gaan, het tot groot belangstelling in die ontwikkeling van alternatiewe bronne gelei. Bio-etanol is 'n goeie alternatief vir fossielbrandstowwe, want dit kan van goedkoop, vollop, hernubare nievoedselbronne geproduseer word. Bio-etanol is ook koolstof-neutraal; die gebruik daarvan laat die netto vlak van koolstofdioksied in die atmosfeer onverstoord. Lignosellulose, en meer spesifiek die sellulose fraksie, is as moontlike grondstof vir die vervaardiging van bio-etanol geïdentifiseer. Die gebruik van lignosellulose as grondstof sal meer volhoubare voorsiening en broodnodige energie-sekuriteit verseker. Sellulose grondstowwe kan in twee hoof kategorieë verdeel word, nl. Newe produkteafval van prosesse anders as brandstofproduksie en gewasse wat spesifiek vir brandstofproduksie gekweek word. Graangewasse soos korog is geïdentifiseer as 'n goeie industriële gewas vir die produksie van energie. Korog se hoër biomassa opbrengs, matige water en voedingstofvereistes, groeiende bewerkingsgebied en die gebruik as 'n veevoergewas eerder as 'n menslike voedselbron, maak dit aantreklik as 'n grondstof vir die vervaardiging van bio-etanol. Die gesamentlike aktiwiteit van endoglukanases, eksoglukanases en β-glukosidases is nodig om kristallyne sellulose tot fermenteerbare suikers te hidroliseer. Die hoë koste van hierdie ensieme is egter die grootste hindernis vir die ekonomiese produksie van bio-etanol vanaf sellulosiese biomassa. 'n Belowende koste verminderingstrategie is die produksie van hierdie sellulolitiese ensieme, die hidrolise van biomassa, en die fermentasie van die suikers na bio-etanol in 'n enkelstap-proses via 'n sellulolitiese mikro-organisme. Die ontwikkeling van so 'n gekonsolideerde bioprosesserings (CBP) organisme kan deur die uitdrukking van sellulolitiese aktiwiteite in 'n nie-sellulolitiese mikro-organisme wat wel in staat is om glukose na etanol om te fermenteer, gerealiseer word. Saccharomyces cerevisiae is 'n goeie kandidaat gasheer vir CBP, omdat hierdie gis ‘n hoë verdraagsaamheid teenoor etanol toon en sy gebruik in industriële toepassings gevestig is. Die ensiematiese aktiwiteite van endoglukanases en eksoglukanases word egter deur die ophoop van sellobiose gedurende die hidrolise van sellulose geïnhibeer. Hierdie effek kan met die byvoeging van meer effektiewe β-glukosidases verlig word. β-Glukosidases hidroliseer sellobiose na glukose en verlig dus die inhibisie op die endoglukanase en eksoglukanase ensiematiese aktiwiteite. Ten spyte van vooruitgang in ensiemproduksie stelsels en ensiemmodifiserings strategieë wat tans vir hoër stabiliteit en aktiwiteit in gebruik is, bestaan daar steeds 'n behoefte om die bestaande versameling van ensieme uit te brei. Bioprospektering vir nuwe sellulolitiese ensieme fokus op spesifieke omgewings, met hoë omsetkoerse van sellulose materiaal of omgewings met uiterste toestande, soos die komposterings-proses. Hierdie ensieme is besig om meer aantreklik in vergelyking met hul mesofieliese eweknieë te raak as gevolg van hul potensiele industriële toepassings en die feit dat hulle die laer natuurlike grense van proteïen-stabiliteit verteenwoordig. / Stellenbosch University and the Technology Innovation Agency for financial support

Extensive research into enzyme-induced bio-conversion of lignocellulose to soluble sugars has been conducted and research continues in this area. Several approaches have been taken to attempt to alleviate the economic problems associated with utilisation of lignocellulose in fuel ethanol production. By expressing cellulase genes in planta, it is hoped that the cost of enzyme-mediated hydrolysis of cellulose to its soluble sugar monomers, will be reduced. Some accomplishments have been made in this area using nuclear genetic transformation (Abdeev et al., 2003; Abdeev et al., 2004; Austin-Phillips et al., 1999; Biswas et al., 2006; Dai et al., 2000a,b; Dai et al., 2005; Jin et al., 2003; Kawazu et al., 1999; Sakka et al., 2000; Ziegelhoffer et al., 1999; Ziegelhoffer et al., 2001; Ziegler et al., 2000), but more research is required to bring the levels of cellulase enzyme expression in plants to levels that will make the process economically competitive. Chloroplasts of N. tabacum were selected as a target for transformation for high level expression due to their extremely high rates of transcription and translation. These were transformed with two genes, the e1 gene from A. cellulolyticus, and the cbh1 gene from T. reesei. Further aims included the investigation of the effects of using different promoters, and the novel use of both nuclear and chloroplast-based expression in a single plant, on the level of protein production in the heterologous host. Heterologous expression of the cbh1 gene was not successful. This is thought to be due to toxicity of the protein in a prokaryotic environment. Future studies should focus on trying to avoid this toxicity by targeting of the chloroplast-expressed enzyme to specific tissues, such as the thylakoid membrane, for containment, creating a codon-optimised synthetic gene that better mimics the codon usage of the plant to be used for expression, or placing the expression under a reactive cascade that is only activated upon exposure to an external trigger. Heterologous expression of the full length gene for E1 from A. cellulolyticus was successful. Chloroplast homology vectors under the constitutive promoter Prrn, and the inducible promoter T7, were constructed and these were used to successfully transform N. tabacum cv. Petit Havana chloroplasts. Stable transgenic plants were produced and evaluated by a variety of means, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 3122 ± 466 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 0.35% ± 0.06 of the total soluble protein. Lastly, chloroplast transformation was combined with nuclear transformation to create novel dual-transgenic plants simultaneously expressing E1 from both the nuclear and chloroplast genomes. The combination of these technologies was very successful, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 35706 ± 955 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 4.78% ± 0.13 of the total soluble protein, and provides a new approach for increasing the accumulation levels of plant-produced cellulase enzymes.