Tesis sobre el tema "Genetic engineering"

2012/2013<br>Machine learning is a suite of techniques that allow developing algorithms for performing tasks by generalizing from examples. Machine learning systems, thus, may automatically synthesize programs from data. This approach is often feasible and cost-effective where manual programming or manual algorithm design is not. In the last decade techniques based on machine learning have spread in a broad range of application domains. In this thesis, we will present several novel applications of a specific machine Learning technique, called Genetic Programming, to a wide set of engineering applications grounded in real world problems. The problems treated in this work range from the automatic synthesis of regular expressions, to the generation of electricity price forecast, to the synthesis of a model for the tracheal pressure in mechanical ventilation. The results demonstrate that Genetic Programming is indeed a suitable tool for solving complex problems of practical interest. Furthermore, several results constitute a significant improvement over the existing state-of-the-art. The main contribution of this thesis is the design and implementation of a framework for the automatic inference of regular expressions from examples based on Genetic Programming. First, we will show the ability of such a framework to cope with the generation of regular expressions for solving text-extraction tasks from examples. We will experimentally assess our proposal comparing our results with previous proposals on a collection of real-world datasets. The results demonstrate a clear superiority of our approach. We have implemented the approach in a web application that has gained considerable interest and has reached peaks of more 10000 daily accesses. Then, we will apply the framework to a popular "regex golf" challenge, a competition for human players that are required to generate the shortest regular expression solving a given set of problems. Our results rank in the top 10 list of human players worldwide and outperform those generated by the only existing algorithm specialized to this purpose. Hence, we will perform an extensive experimental evaluation in order to compare our proposal to the state-of-the-art proposal in a very close and long-established research field: the generation of a Deterministic Finite Automata (DFA) from a labelled set of examples. Our results demonstrate that the existing state-of-the-art in DFA learning is not suitable for text extraction tasks. We will also show a variant of our framework designed for solving text processing tasks of the search-and-replace form. A common way to automate search-and-replace is to describe the region to be modified and the desired changes through a regular expression and a replacement expression. We will propose a solution to automatically produce both those expressions based only on examples provided by user. We will experimentally assess our proposal on real-word search-and-replace tasks. The results indicate that our proposal is indeed feasible. Finally, we will study the applicability of our framework to the generation of schema based on a sample of the eXtensible Markup Language documents. The eXtensible Markup Language documents are largely used in machine-to-machine interactions and such interactions often require that some constraints are applied to the contents of the documents. These constraints are usually specified in a separate document which is often unavailable or missing. In order to generate a missing schema, we will apply and will evaluate experimentally our framework to solve this problem. In the final part of this thesis we will describe two significant applications from different domains. We will describe a forecasting system for producing estimates of the next day electricity price. The system is based on a combination of a predictor based on Genetic Programming and a classifier based on Neural Networks. Key feature of this system is the ability of handling outliers-i.e., values rarely seen during the learning phase. We will compare our results with a challenging baseline representative of the state-of-the-art. We will show that our proposal exhibits smaller prediction error than the baseline. Finally, we will move to a biomedical problem: estimating tracheal pressure in a patient treated with high-frequency percussive ventilation. High-frequency percussive ventilation is a new and promising non-conventional mechanical ventilatory strategy. In order to avoid barotrauma and volutrauma in patience, the pressure of air insufflated must be monitored carefully. Since measuring the tracheal pressure is difficult, a model for accurately estimating the tracheal pressure is required. We will propose a synthesis of such model by means of Genetic Programming and we will compare our results with the state-of-the-art.<br>XXVI Ciclo<br>1984

Bibliography: leaves 127-151. The successful application of genetic engineering in wheat is dependent on the availability of suitable tissue culture and transformation methods. The primary object of this project was the development of these technologies using elite Australian wheat varieties.

Dissertation (PhD)--University of Stellenbosch, 2005.<br>ENGLISH ABSTRACT: To manipulate recombinant microorganisms for industrial processes, controllable genetic systems are needed that can coordinate expression of recombinant metabolic pathways. All components are sensitive to change and thus putative targets for modification and genetic elements and regulatory systems need to be understood and determined. Central in gene regulation is the transcription activators that mediate gene transcription mechanisms by binding to promoters in response to environmental signals. Promoter engineering entails the modification of transcription factors and their target promoters. In this study, a metabolic control system in Saccharomyces cerevisiae was constructed that would allow induction in response to physiological environment, specifically hypoxia and low temperature conditions. Two approaches were undertaken to find such a system. Firstly, a bi-directional reporter gene cloning vector was designed to search for novel hypoxiainducible promoters. Secondly, a transcription regulatory circuit was built, consisting of an inducible transcription regulator and promoter with a reporter gene through which it mediates transcription. Advantage was taken of the modular nature of proteins and functional domains originating from different transcriptional proteins were combined. A search for promoter elements sensitive to hypoxia from a S. cerevisiae genomic DNA (gDNA) library, using a bi-directional cloning vector, did not yield highly inducible promoters. It was concluded that a multitude of signals overlap, rendering genetic induction difficult to control. A synthetic regulatory system would minimize the impact of these multiple interactions. Such a genetic circuit was constructed, consisting of a chimeric transcription activator and a target fusion promoter. The chimeric transcription activator consisted of the GAL4 DNA binding domain, ADR1 TADIII transactivation domain and three domains of the MGA2 regulatory protein. The functional domains of Mga2p responsible for unregulated expression (at high basal levels) under both aerobic and hypoxia conditions were located, as well as a further upregulation under low temperature, and were mapped to the Nterminal and mid-Mga2p regions. A target fusion promoter consisting of a partial GAL10/1 promoter sequence and a Trichoderma reesei core xyn2 promoter were constructed as target for this chimeric transactivator. This synthetic promoter was fused to the T. reesei xyn2 open reading frame encoding for a readily assayable β-xylanase activity. Both the chimeric transactivator and fusion promoter-reporter gene cassettes were expressed from the same episomal plasmid, named pAR. Transformed into S. cerevisiae Y294, this regulatory system induced transcription under aerobic and hypoxia conditions. Furthermore, the reporter gene expression was upregulated by the chimeric transactivator at low temperatures. The chimeric transactivator mediated a seven-fold induction of the reporter gene under aerobic conditions in S. cerevisiae Y294 when transformed with plasmid AR. A two- to three-fold induction at 23ºC was reported under anaerobic conditions, relative to a reference strain expressing a transcription activator without the Mga2p domains. At 30ºC, a two- to three-fold induction under aerobic conditions and similar induction under oxygen-limited conditions were observed. Replacing the reporter gene with your favorite gene (for example a recombinant enzyme) and incorporating such a pAR system into a recombinant yeast should induce expression of the chosen gene under low temperatures, both aerobic and anaerobically (thus creating a controllable system). The system also has wider application in identifying other transcription factors’ signal-sensitive domains. The design of this system provides the ability to add a linker to a transactivator and to either create specific signal sensitivity or relieve the regulator of its signal dependence. It creates an easy system for assessing other transactivators and their domains with unknown functions and thus provides a ”workhorse and prospector in one”.<br>AFRIKAANSE OPSOMMING: Vir die manipulering van rekombinante mikroörganismes vir industriële prosesse word beheerbare genetiese stelsels benodig om gekoördineerde uitdrukking van rekombinante metaboliese weë teweeg te bring. Alle komponente van sulke stelsels is sensitief vir verandering en genetiese elemente en reguleerbare sisteme moet dus deeglik verstaan of bepaal word. Sentraal tot geenregulering is die transkripsie-aktiveerders wat geentranskripsie beheer deur aan promoters te bind in reaksie op eksterne omgewingsfaktore. Promotoringenieurswese behels wysigings van transkripsiefaktore en hul teikenpromotors. In hierdie studie is 'n genetiese beheerstelsel vir Saccaromyces cerevisiae ontwikkel wat induksie in reaksie tot spesifieke fisiologiese omgewingreaksies, naamlik hipoksie- en lae temperatuur, toelaat. Twee benaderings is gevolg: eerstens is ‘n tweerigting verklikker-geen vektor ontwikkel en gebruik om vir unieke induseerbare hipoksie-promoters te soek. Tweedens is ‘n transkripsie reguleringstelsel gebou wat uit ‘n induseerbare transkripsiereguleerder and promotor met ‘n verklikkergeen bestaan, waardeur transkripsie bemiddel kan word. Hierdie benadering benut die modulêre onderbou van proteïene en funksionele domeine afkomstig vanaf verskillende transkripsiefaktore is gekombineer. 'n Soektog na hipoksie-sensitiewe promotors vanuit 'n Saccharomyces cerevisiae-genoom- DNA (gDNA), deur van ‘n tweerigting verklikker-vektor gebruik te maak, het ongelukkig nie hoogs-induseerbare promotors opgelewer nie. Die gevolgtrekking was dat ‘n veelvoud van seine met mekaar oorvleuel en die beheer van genetiese induksie dus bemoeilik. Die ontwikkeling van ‘n sintetiese regulering-sisteem kan die impak van die veelvuldige interaksies verminder. Vir dié doel is ‘n sintetiese reguleringstelsel ontwerp, bestaande uit ‘n chimeriese transkripsie-aktiveerder met ‘n teiken fusie-promotor. Die chimeriese transaktiveerder bestaan uit die GAL4 DNA bindingsdomein, die ADR1 TAD III transaktiveringsdomein en drie domeine van die Mga2 reguleringsproteïen. In die studie is die funksionele domeins van Mga2p betrokke by lae temperatuur-respons en ongereguleerde uitdrukking (teen hoë basale vlakke) onder beide aërobiese en anaërobiese toestande aangedui en is tot die N-terminaal en middel-Mga2p areas gekarteer. ‘n Teiken-fusie-promoter, bestaande uit 'n gedeeltelike GAL1/10 DNA promotoropeenvolging en ‘n Trichoderma reesei kern xyn2-promoter, is as teiken vir hierdie chimeriese transaktiveerder saamgestel. Hierdie sintetiese promotor is aan die T. reesei xyn2 oopleesraam, wat vir ‘n maklik meetbare β-xylanase aktiwiteit kodeer, gekoppel. Beide die chimeriese transaktiveerder and fusie-promoter-verklikker-geenkaset word vanaf dieselfde episomale plasmied, bekend as pAR, uitgedruk. Hierdie reguleringsisteem induseer transkripsie onder aërobiese en hipoksie toestande in S. cerevisiae Y294. Verder word die verklikkergeen se uitdrukking deur die chimeriese transaktiveerder by lae temperature verhoog. Die chimeriese transaktiveerder induseer ‘n sewe-voudige induksie van die verklikkergeen onder aërobiese toestande by 23ºC vanaf die pAR-stelsel in S. cerevisiae Y294. ‘n Twee- tot drie-voudige induksie teen 23ºC is onder hipoksie toestande gevind, relatief tot induksievlakke van ‘n verwysingstam met ‘n transaktiveerder sonder die Mga2 domeine. By 30ºC is ‘n twee- tot drie-voudige induksie onder aërobiese en lae suurstofvlakke waargeneem. Deur die verklikker geen met ‘n jou-gunsteling-geen te vervang (bv. ‘n rekombinante ensiem) en so 'n pAR-sisteem in ‘n rekombinante gis te inkorporeer, word uitdrukking onder lae temperature onder beide aërobiese- en anaërobiese toestande geïnduseer (en sodoende word ‘n reguleerbare sisteem geskep). Die sisteem het wyer toepassing om sein-sensitiewe domeine van ander transkripsiefaktore te identifiseer. Die ontwerp van die stelsel maak dit moontlik om 'n skakel tot die transaktiveerder by te voeg wat óf sensitiwiteit tot 'n spesifieke sein skep, óf die reguleerder vanaf seinafhanklikheid verlos. So word ‘n bruikbare stelsel vir die bestudering van ander transaktivators en hul domeine met onbekende funksie geskep – ‘n “werksesel en prospekteerder in een”.

This thesis aims to provide a more critical framework for the assessment of future technologies and therefore social directions and to help to bring an understanding to the relationship between global political economy, corporate power, ideology, science and technology. This is essential given the many issues facing contemporary society – issues of sustainability and humanity’s place in the broad ecology, of the need for a diversity of economies, societies and cultures, of the need for greater economic equality and equity across the globe. The relationship between globalisation, science and technology, democratic governance and citizens is explored using the case of genetic engineering technologies. The thesis draws on a conceptual framework provided by the theory of political economy to facilitate the assessment of the impact of a technology on society . It provides a critical framework for looking at individualised, sectoral and short term interests versus the often conflicting interests of what is termed the ‘common good’. The juxtaposition of the neo-liberal, conservative and contemporarily dominant world view with that of the more radical, political economy stance exposes the tension between these two ways of viewing human history and the future of humankind.

Particle bombardment of immature scutella was used to generate multiple lines of transgenic wheat with either PHY A, PHYB or PHYC from Arabidopsis or PHY A from oat.;Responses of segregating transgenic seedlings to cFR light were used as a screen for biologically active lines. The 'short coleoptile' phenotype under cFR was shown to segregate with the oat PHYA transgene and rtPCR detection of transcript. All of the transgenic lines containing the Arabidopsis PHYA and several oat PHYA lines demonstrated phenotypes indistinct to wild-type, possibly demonstrating transcriptional silencing.;Biologically active lines with Mendelian segregation ratios of 3:1 were selected for further analysis and homozygous individuals for each identified by PCR. Southern analysis of selected lines demonstrated that all originated from a distinct transformation event and harboured at least a single intact copy of the transgene. The complexity of gene integration was related to observed gene dosage and silencing effects.;Adult transgenic plants under white light with high R:FR ratio, demonstrated an increased chlorophyll content, a reduced production of superfluous tillers, a reduction in sensitivity to shortened day-length and an increased developmental rate leading to a reduction in days to heading.;The transgenic lines demonstrated an altered response to shade. In addition to the phenotypes observed under white light, plant height was significantly reduced and harvest index increased compared to the nulls, these responses were distinct to the those observed under white light and suggest an altered response to reduced R:FR.;The results suggest that oat PHYA is biologically active in transgenic wheat and that over-expressing PHYA alters the responses of etiolated seedlings to cFR, the light grown morphology and development of adult plants, and the responses of the plants to shade.

Modern medical science and the needs of practical health care require the use of scientific technology in basic research. The Europian federation of Biotechnology defines modern biotechnology as the use of sciences related to nature (biology, chemistry, physics) and engineering (e.g. electronics) in relation with biosystems in the bio-industry.

"Template" and "adder" proteins can be genetically encoded to produce large amyloid fibers when mixed together. Escherichia coli is used to clone a "template" protein, Gd20, which will cooperatively self-assemble with two "adder" proteins, P7Q and P7S, to yield two different large amyloid fibers. Atomic force microscopy (AFM) is used to image the fibers and AFM tip approach/retraction force is used to quantify molecular packing in the fibers. Glutamine (Q)-containing P7Q and serine (S)-containing P7S both have the same hydrophobic core, charge, and hydrogen bonding potential. However, P7Q is highly alpha-helical while P7S contains a beta-sheet core. After 72 hours, the Gd20:P7Q template:adder protein mixture produces tightly packed ~0.3 μm high and ~1.9 μm wide fibers that exhibit a low retraction force of ~44 nN after indentation. The Gd20:P7S mixture produces larger ~1.1 μm high and ~9.7 μm wide fibers exhibiting a much higher retraction force of ~503 nN showing they are much less molecularly packed. These results indicate that the adder protein alpha-helical character is important for self-assembly and molecular packing inside of the large amyloid fiber. The experimental results show that large amyloid fibers with predictable size and mechanical properties can be anticipated and encoded at the genetic level.<br>Master of Science

This thesis aims to provide a more critical framework for the assessment of future technologies and therefore social directions and to help to bring an understanding to the relationship between global political economy, corporate power, ideology, science and technology. This is essential given the many issues facing contemporary society - issues of sustainability and humanity's place in the broad ecology, of the need for a diversity of economies, societies and cultures, of the need for greater economic equality and equity across the globe. The relationship between globalisation, science and technology, democratic governance and citizens is explored using the case of genetic engineering technologies. The thesis draws on a conceptual framework provided by the theory of political economy to facilitate the assessment of the impact of a technology on society. It provides a critical framework for looking at individualised, sectoral and short term interests versus the often conflicting interests of what is termed the 'common good'. The juxtaposition of the neo-liberal, conservative and contemporarily dominant world view with that of the more radical, political economy stance exposes the tension between these two ways of viewing human history and the future of humankind.

This thesis aims to provide a more critical framework for the assessment of future technologies and therefore social directions and to help to bring an understanding to the relationship between global political economy, corporate power, ideology, science and technology. This is essential given the many issues facing contemporary society - issues of sustainability and humanity's place in the broad ecology, of the need for a diversity of economies, societies and cultures, of the need for greater economic equality and equity across the globe. The relationship between globalisation, science and technology, democratic governance and citizens is explored using the case of genetic engineering technologies. The thesis draws on a conceptual framework provided by the theory of political economy to facilitate the assessment of the impact of a technology on society. It provides a critical framework for looking at individualised, sectoral and short term interests versus the often conflicting interests of what is termed the 'common good'. The juxtaposition of the neo-liberal, conservative and contemporarily dominant world view with that of the more radical, political economy stance exposes the tension between these two ways of viewing human history and the future of humankind.

The development of processes for sustainable energy and chemical production is of great importance for the health of our planet. Utilising suitable feedstocks such as renewable resources and existing waste streams is central to making such processes a reality. Microorganisms can be employed in the processing of a diverse range of such feedstocks, offering unique routes of production for useful and valuable chemical products. Acetogenic organisms, capable of fermenting single carbon (C1) feedstocks are especially interesting from the perspective of industrial application. Their natural metabolism and biochemistry enables fixation of low energy C1 compounds, under conditions which would typically be unfeasible with traditional chemical catalysts. Genetic research and development of new acetogenic species opens the door to industrially feasible, and economically attractive, sustainable bioprocessing. This study outlines the genetic development of the methanol and syngas fermenting acetogen E. limosum. This includes establishing gene-transfer and genetic engineering tools in this organism for first time. Additionally, we demonstrate the genetic engineering of synthetic metabolic pathways in this strain to enable production of valuable chemicals, acetone and isopropanol. Gene-transfer is a cornerstone of modern genetic research in microorganisms, and so effective methods of establishing it are of significant value. We present the development of an improved methodology for enabling and enhancing gene transfer in recalcitrant microorganisms which contain active restriction modification (RM) systems. The method harnesses lambda-red recombineering to support the rapid creation of tailored methylation donor (MD) strains for the preparation and protection of transforming plasmids. The process is uniquely designed in a manner which enables compatibility with both electroporation and conjugation methods of gene transfer.

New technologies are making it possible to engineer organisms with fundamentally new and useful properties. In vivo genome engineering technologies capable of manipulating genomes from the nucleotide to the megabase scale were developed and applied to reassign the genetic code of Escherichia coli. Such genomically recoded organisms show promise for thwarting horizontal gene transfer with natural organisms, resisting viral infection, and expanding the chemical properties of proteins.

Thesis (M.S.)--East Tennessee State University, 2003.<br>Title from electronic submission form. ETSU ETD database URN: etd-1103103-095304. Includes bibliographical references. Also available via Internet at the UMI web site.

Bacillus anthracis is a Gram positive, spore forming, non-motile, rod shaped, soil bacterium, and is endemic worldwide. Currently, the biology of B. anthracis is poorly understood. B. anthracis is one of many biological weapons used today. A -/- mutant strain of B. anthracis that lacks the pathogenic plasmids was created by serial culture at 42°C. Key DNA replication genes were identified by homology as targets. The dnaB gene, essential for B. subtilis initiation of DNA replication, was my focus. A vector system was created by polymerase chain reaction (PCR) with the pMUTIN4 integration vector and the promoter region of dnaB to study the genetics of B. anthracis. An electro-transformation system was formulated to knock-out the -/- B. anthracis dnaB gene. We have successfully incorporated the pMUTIN4 vector into the chromosomal DNA of B. anthracis. We also have formulated an electro-transformation system and vector system for use in B. anthracis.

Thesis (S.B. and M.Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1999.<br>Includes bibliographical references (leaves 50-51).<br>by Jason C. Miller.<br>S.B.and M.Eng.

There are many technologies being developed for crop breeding. Two interesting technologies are genetic modification and genetic editing. Competitive pressures and changing consumer preferences are forcing organizations to invest heavily in these two technologies. Organizations must decide which traits they want to target and must commit significant time a money to the project. Traditionally, firms would decide which project to embark on if the project is net present value positive. Throughout the research and development process managers have flexibility to abandon the project once new information is received. That flexibility has value and real option analysis must be performed to value that flexibility. Once the value of a GM and GE project is determined, how might an organization decide which project to do? The concept of minimum required acreage (MRA) is developed in this study, allowing organizations to compare GM and GE technologies and decide which project to invest it.

Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.

Genetic algorithms (GAs) are stochastic search techniques inspired by the principles of natural selection and natural genetics which have revealed a number of characteristics particularly useful for applications in optimization, engineering, and computer science, among other fields. In control engineering, they have found application mainly in problems involving functions difficult to characterize mathematically or known to present difficulties to more conventional numerical optimizers, as well as problems involving non-numeric and mixed-type variables. In addition, they exhibit a large degree of parallelism, making it possible to effectively exploit the computing power made available through parallel processing. Despite their early recognized potential for multiobjective optimization (almost all engineering problems involve multiple, often conflicting objectives), genetic algorithms have, for the most part, been applied to aggregations of the objectives in a single-objective fashion, like conventional optimizers. Although alternative approaches based on the notion of Pareto-dominance have been suggested, multiobjective optimization with genetic algorithms has received comparatively little attention in the literature. In this work, multiobjective optimization with genetic algorithms is reinterpreted as a sequence of decision making problems interleaved with search steps, in order to accommodate previous work in the field. A unified approach to multiple objective and constraint handling with genetic algorithms is then developed from a decision making perspective and characterized, with application to control system design in mind. Related genetic algorithm issues, such as the ability to maintain diverse solutions along the trade-off surface and responsiveness to on-line changes in decision policy, are also considered. The application of the multiobjective GA to three realistic problems in optimal controller design and non-linear system identification demonstrates the ability of the approach to concurrently produce many good compromise solutions in a single run, while making use of any preference information interactively supplied by a human decision maker. The generality of the approach is made clear by the very different nature of the two classes of problems considered.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2005.<br>Biomass is the sole foreseeable sustainable source of organic fuels, chemicals and materials. It is a rich and renewable energy source, which is abundant and readily available. Primary factors motivating the use of renewable enrgy sources include the growing concern over global climate change and the drastic depletion of non-renewable resources. Among various forms of biomass, cellulosic feedstocks have the greatest potential for energy production from. The biggest technological obstacle to large-scale utilisation of cellulosic feedstocks for the production of bioethanol as a cost-effective alternative to fossil fuels is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass. A promising strategy to overcome this impediment involves the production of cellulolytic enzymes, hydrolysis of biomass and fermentation of resulting sugars to ethanol in a single process step via a single microorganism or consortium. Such “consolidated bioprocessing” (CBP) offers very large cost reductions if microorganisms, such as the yeast Saccharomyces cerevisiae, can be developed that possess the required combination of efficient cellulose utilisation and high ethanol yields. Cellulose degradation in nature occurs in concert with a large group of bacteria and fungi. Cellulolytic microorganisms produce a battery of enzyme systems called cellulases. Most cellulases have a conserved tripartite structure with a large catalytic core domain linked by an O-glycosylated peptide to a cellulose-binding domain (CBD) that is required for the interaction with crystalline cellulose. The CBD plays a fundamental role in cellulose hydrolysis by mediating the binding of the cellulases to the substrate. This reduces the dilution effect of the enzyme at the substrate surface, possibly by helping to loosen individual cellulose chains from the cellulose surface prior to hydrolysis. Most information on the role of CBDs has been obtained from their removal, domain exchange, site-directed mutagenesis or the artificial addition of the CBD. It thus seems that the CBDs are interchangeable to a certain degree, but much more data are needed on different catalytic domain-CBD combinations to elucidate the exact functional role of the CBDs. In addition, the shortening, lengthening or deletion of the linker region between the CBD and the catalytic domain also affects the enzymatic activity of different cellulases. Enzymes such as the S. cerevisiae exoglucanases, namely EXG1 and SSG1, and the Saccharomycopsis fibuligera β-glucosidase (BGL1) do not exhibit the same architectural domain organisation as shown by most of the other fungal or bacterial cellulases. EXG1 and SSG1 display β-1,3-exoglucanase activities as their major activity and exhibit a significant β- 1,4-exoglucanase side activity on disaccharide substrates such as cellobiose, releasing a free glucose moiety. The BGL1 enzyme, on the other hand, displays β-1,4-exoglucanase activity on disaccharides. In this study, the domain engineering of EXG1, SSG1 and BGL1 was performed to link these enzymes to the CBD2 domain of the Trichoderma reesei CBHII cellobiohydrolase to investigate whether the CBD would be able to modulate these non-cellulolytic domains to function in cellulose hydrolysis. The engineered enzymes were constructed to display different modular organisations with the CBD, either at the N terminus or the C terminus, in single or double copy, with or without the synthetic linker peptide, to mimic the multi-domain organisation displayed by cellulases from other microorganisms. The organisation of the CBD in these recombinant enzymes resulted in enhanced substrate affinity, molecular flexibility and synergistic activity thereby improving their ability to act and hydrolyse cellulosic substrates, as characterised by adsorption, kinetics, thermostability and scanning electron microscopic (SEM) analysis. The chimeric enzyme of CBD2-BGL1 was also used as a reporter system for the development and efficient screening of mutagenised S. cerevisiae strains that overexpress CBD-associated enzymes such as T. reesei cellobiohydrolase (CBH2). A mutant strain WM91 was isolated showing up to 3-fold more cellobiohydrolase activity than that of the parent strain. The increase in the enzyme activity in the mutant strain was found to be associated with the increase in the mRNA expression levels. The CBH2 enzyme purified from the mutant strain did not show a significant difference in its characteristic properties in comparison to that of the parent strain. In summary, this research has paved the way for the improvement of the efficiency of the endogenous glucanases of S. cerevisiae, and the expression of heterologous cellulases in a hypersecreting mutant of S. cerevisiae. However, this work does not claim to advance the field closer to the goal of one-step cellulose processing in the sense of technological enablement; rather, its significance hinges on the fact that this study has resulted in progress towards laying the foundation for the possible development of efficient cellulolytic S. cerevisiae strains that could eventually be optimised for the one-step bioconversion of cellulosic materials to bioethanol.

In the next fifty years world population will reach 9-10 billion of people. This increment will drastically increase energy and food demand. The current global food and energy supply chain is not sustainable and it causes increasing CO2 emissions, exacerbating the greenhouse effect and the climate change, whose effects we are already experiencing. The Climate Change conference hold in Paris in 2015 showed a global consensus on the need to drastically reduce carbon emissions to avoid the environmental disaster, which is leading to many negative effects like desertification, animal and plants species extinction and ocean acidification. In order to face this challenge different strategies are under study and development. Microalgae are emerging as an interesting possibility for the production of energy and food. Being photosynthetic organisms, algae biomass is produced from CO2 fixation, and contains proteins, and lipids exploitable as food or fuel. Despite their potential, microalgae production on a large scale is still not competitive on the food and energy market. One of the main limitation is that photosynthetic efficiency at the industrial scale is reduced, negatively affecting growth and biomass accumulation. All first attempts of algae large scale cultivation have been pursued cultivating wild type strains (WT) that evolved in an environment extremely different and thus they are not adapted to have a maximal productivity in the industrial system. As done with crops domestication, there is the need to modify genetically strains in order to adapt them to grow in industrial systems and increase productivity. There are different algal species that are emerging as promising candidates for food and fuel production and among them the largest part of this work is focused on Nannochloropsis gaditana, a marine microalga able to accumulate lipids and molecules like β-carotene interesting for nutraceutical purposes. After a general introduction, chapter 2 described the generation of a collection of random mutants of N.gaditana exploiting two different mutagenic approaches, one based on the use of the mutagen compound Ethyl Methane Sulfonate (EMS) and the other one based on random insertion of a resistance cassette to zeocin. The collection was screened for strains altered in the photosynthetic apparatus using a multiple steps in vivo fluorescence analysis. In Appendix 1 I collaborated in evaluation of the productivity for one of these strains (E2) in a fed batch system mimicking an industrial culture. E2 was selected for a reduction in the chlorophyll content and also in the PSII antenna size. One of the limitations in algae mass cultures is that light penetration negatively affects growth and thus a strain with a reduction in the light absorption capacity could have a productivity advantage. This work indeed showed that the strain E2 had increased productivity with respect to WT, but also highlighted the seminal influence of the cultivation parameters on strains performances. Most of the PhD work was then invested on the characterization of two promising insertional strains. The first strain presented in chapter 3 is I29. This is a low chlorophyll (Chl) content mutant with no difference in the antenna size of PSII. In flask culture I29 showed a reduction of 20% in the Chl content respect to the WT, which leaded also to a higher electron transport rate (ETR). The site of insertion of the resistance cassette was identified but there was no apparent effect on transcription level of closest genes. The full genome of the strain was thus re-sequenced revealing a point mutation in a key gene involved in the chlorophyll biosynthetic pathway, a highly likely candidate to justify the phenotype. I29 in lab scale PBR was also assessed evidencing an increase in productivity of 14% with respect to the WT. An analogous work was done in chapter 4 for the characterization of another interesting mutant from the collection isolated in chapter 2, the mutant I48. This mutant has a severe reduction in the non-photochemical quenching (NPQ) activation. The site of insertion was not identifiable because of the presence of tandem insertions and again the strain genome was re-sequenced highlighting the presence of point mutations due to electroporation. Among these we found an interesting one in the gene codifying for the protein LHCX1. This is a stress-related antenna protein which in other species like Phaeodactylum tricornutum has already been recognized as a key component of the fast NPQ response. The loss of the protein accumulation was confirmed by Western Blot. Strain response to different illumination conditions was also evaluated highlighting the ability of the mutant to grow also in high light condition. The last part of the characterization was devoted to productivity evaluation since a depletion of NPQ could be advantageous in an industrial condition of a dense culture, where only the external layer are exposed to intense light. A low NPQ phenotype can avoid undesired energy dissipation in the internal layers. We observed a 24% higher productivity for I48 respect to the WT but, as discussed in Appendix 1 this was dependent from growing conditions. In diatoms NPQ has been associated not only to the high light response but also to the fluctuating light (FL) response, which is a condition easily experienced in outdoor cultivation system. We take advantage from the isolation of this mutant with a severe reduction in NPQ to verify what can be the impact of NPQ in N.gaditana response. In chapter 5 we set up an experiment in which we exposed the cultures to 150 μmol of photons m-2s-1 but in the control sample the light intensity was constant, in FLs it was the result of a combination of a low light exposition with high light flashes given with different frequencies and duration. We found that those FL treatments caused a severe growth reduction in WT but I48 showed no increased sensitivity. The growth reduction was proportional to the flash frequency and stronger with higher flash frequency. An analysis of photosynthetic apparatus functionality evidenced that in FL PSII activity is not affected while PSI is largely inactivated. This suggests that alternatives electron transport around PSI, that are active in avoiding its over-excitation, are not as efficient in N.gaditana as in other organisms. These first 5 chapters highlight the potential of microalgae engineering to find new strains more productive respect to the WT. FL experiment not only adds new knowledge about NPQ role in N.gaditana, but taking in count that FL is a condition easily found if we want to exploits sunlight in industrial system, it can be the starting point to find new gene targets to improve productivity. This work started with an insertional approach that in principle should facilitate identification of the genes responsible of the phenotype. In this case this advantage is impaired by the frequent presence of tandem insertions and accumulation of point mutations during transformation. Alternative strategies are thus more suitable and will be pursued in the future. In chapter 6 we tested the chemical mutagenesis and the screening method set up for N.gaditana with Scenedesmus obliquus to evaluate if the approach could be generally applied to any species of interest. Indeed at least a strain with interesting properties was isolated (SOB17). In this work we also evaluated productivity in a flat panel in continuous mode. Indeed the geometry of a flat panel leads to a better light distribution and homogeneity respect to other system like the bottles. Moreover the continuous mode provides a stable culture continuously producing. This culture showed remarkable stability for more than 30 weeks and SOB17 showed a higher productivity in one of the conditions tested but, as observed for Nannochloropsis gaditana strains also in this case operational conditions were shown to have a fundamental influence on strains performances. In addition to the Appendix 1 described above, other two appendix sections are also included in the thesis. They are the results of two different collaboration in which some of the experimental techniques exploited in this work were applied with different aims. In Appendix II, there is the published work “Genetic Engineering of algae photosynthetic productivity using mathematical models” that describes the development of a mathematical model to evaluate the key factors influencing algae biomass productivity in PBR. The model was shown to be able to predict the effect of genetic modifications on algae performances in an industrial context, thus providing a valuable tool to identify the genotypes with the best advantages for productivity. In Appendix III is reported the collaboration with Prof. Angela Falciatore (Diatom Functional Genomics team, at the UPMC-Paris). A screening procedure similar to those one set up for N.gaditana was adapted to search photosynthetic alterations in a collection of Phaeodactylum tricornutum transgenic lines genetically modified by the RNA interference approach, in order to modulate the expression of transcription factors (TF). Thank to this screening procedure we found some promising correlation between the genetic modulation of a class of TFs and the photosynthetic phenotype of mutants strains isolated. Now this correlation phenotype-TF expression level is under validation by confirming the screening phenotype and evaluating the transcripts level.<br>Si stima che nei prossimi 50 anni la popolazione mondiale aumenterà fino a raggiungere i 9-10 miliardi di persone. Un incremento di queste proporzioni porterà inevitabilmente ad una maggior richiesta di produzione di cibo ed energia. L’attuale metodo di approvvigionamento di queste risorse non è sostenibile in quanto è la causa principale dell’aumento delle emissioni di CO2 in atmosfera, le quali stanno alimentando il cambiamento climatico. Nella conferenza che si è tenuta a Parigi nel 2015 sul cambiamento climatico è emersa chiaramente la necessità di un cambio di tendenza per poter evitare il collasso ambientale, che sta già portando alla desertificazione di molti terreni un tempo fertili, all’estinzione di massa di specie animali e vegetali e all’acidificazione degli oceani. Per invertire questa tendenza quindi bisogna rivedere il nostro sistema di produzione alimentare ed energetica. Tra le possibilità al vaglio le microalghe stanno emergendo come un interessante candidato sia per la produzione di energia che di cibo. Infatti le microalghe sono organismi fotosintetici che producono biomassa fissando CO2. Molte specie sono ricche di proteine e lipidi utilizzabili come nutrimento o come matrice per la produzione di biodiesel. Tuttavia nonostante le loro potenzialità le microalghe non sono ancora utilizzate su larga scala perché la loro produzione continua ad aver un costo troppo elevato a fronte di produttività insufficienti. Uno dei maggiori limiti è legato al calo di efficienza fotosintetica che si registra nel passaggio in scala industriale, la quale impatta negativamente sulla crescita delle microalghe e quindi sulla produzione di biomassa. Tuttavia bisogna tenere conto che ad ora si stanno coltivando ceppi wild type, che quindi si sono evoluti ed adattati per crescere al meglio in un ambiente naturale estremamente diverso da quello industriale. Perciò come si è fatto con la domesticazione delle piante per uso alimentare, sarà necessario un passaggio di ingegnerizzazione dei ceppi parentali per ottenere dei nuovi ceppi più adatti alle condizioni industriali e quindi più produttivi una volta coltivati su larga scala. Ci sono svariate specie algali al vaglio come candidati per la produzione alimentare ed energetica, ma questo lavoro si focalizza principalmente su Nannochloropsis gaditana. Questa è una microalga marina capace di accumulare sia lipidi sia molecole di interesse per la nutraceutica come il β-carotene. Dopo una introduzione generale, nel capitolo 2 viene descritto l’ottenimento di una collezione di mutanti casuali generati attraverso due diversi approcci di mutagenesi. Il primo è un approccio di mutagenesi chimica, che utilizza come agente mutageno l’etil-metano-sulfonato (EMS) e il secondo è un approccio di mutagenesi inserzionale che sfrutta l’inserimento casuale nel genoma di DNA esogeno che conferisca al ceppo la resistenza per la zeocina. Da questa collezione si sono isolati solo i mutanti alterati nell’apparato fotosintetico utilizzando cicli progressivi di selezione attraverso misure di fluorescenza in vivo. In Appendice 1 è riportata la collaborazione nella valutazione delle produttività di uno dei mutanti ottenuti con EMS (E2), in condizioni di semi continuo che mimano una coltura industriale. E2 è stato selezionato perché presentava un ridotto contenuto di clorofilla abbinato anche ad una riduzione del numero di proteine antenna legate al PSII. Infatti uno dei maggiori problemi su scala industriale è la scarsa penetrazione della luce, dovuta alle densità elevate raggiunte dalle colture, la quale ha un impatto negativo sulla produzione. Un mutante con una ridotta capacità di assorbimento della luce può essere vantaggioso perché migliora la distribuzione della luce nel sistema con un conseguente incremento nella crescita. Questo lavoro infatti dimostra come E2 sia più produttivo del WT, ma sottolinea anche che le condizioni di coltura sono fondamentali per utilizzare al meglio il potenziale del ceppo mutato. La maggior parte di questa tesi è stata poi dedicata alla caratterizzazione di due ceppi promettenti ottenuti per mutagenesi inserzionale. Il primo è I29 ed è presentato nel capitolo 3. Questo mutante è stato selezionato perché presentava un ridotto contenuto di clorofilla anche se il numero di proteine antenna per PSII è analogo al WT. In beuta la coltura di I29 mostra un 20% in meno di clorofilla rispetto al WT, che si accompagna con un trasporto elettronico che si satura ad intensità di luce maggiori (maggiore ETR electron transport rate). Il sito di inserzione della cassetta è stato identificato come singolo, ma sembra non influenzare i livelli di trascrizione dei geni vicini. Il completo ri-sequenziamento del genoma ha evidenziato una mutazione puntiforme in un gene chiave nella biosintesi della clorofilla, che può essere un promettente candidato per giustificare il fenotipo di I29. Di questo mutante si è valuta anche la produttività e si è visto un incremento del 14% rispetto al WT. Un lavoro analogo è stato fatto per il mutante I48, isolato sempre nel capitolo 2. Questo mutante ha una importante riduzione nell’attivazione dei meccanismi di dissipazione dell’eccesso di energia luminosa come calore (NPQ). Non è stato possibile individuare il sito di inserzione a causa della presenza di inserzioni multiple in tandem, perciò anche in questo caso il genoma è stato ri-sequenziato. Il sequenziamento ha evidenziato nuovamente la presenza di mutazioni puntiformi dovute al protocollo di trasformazione. Tra queste è emersa una mutazione nel gene codificante per la proteina LHCX1. Questa è una proteina antenna coinvolta nella risposta allo stress luminoso, il cui ruolo chiave nella risposta rapida del NPQ è già stato evidenziato in altre specie come Phaeodactylum tricornutum. Il mancato accumulo di questa proteina è stato confermato attraverso Western Blot. Si è valutata la capacità di questo ceppo di crescere in diverse condizioni luminose. È emerso che I48 è in grado di crescere anche in alta luce nonostante questa riduzione nella risposta di NPQ. L’ultima parte della caratterizzazione ne ha valutato invece la produttività. Infatti una riduzione nel NPQ può essere vantaggiosa in condizioni industriali in quanto in una coltura densa solo lo strato esterno è esposto a luci intense. Perciò un NPQ ridotto può evitare una dissipazione indesiderata di energia negli strati più interni dove la luce è già limitante. I48 ha mostrato un aumento di produttività del 24% rispetto al WT, ma come già discusso in Appendice 1 questo è strettamente dipendente dalle condizioni di coltura. Nelle diatomee il NPQ è stato associato non solo alla risposta ad alta luce ma anche a quella alla luce fluttuante (FL), una condizione facilmente sperimentabile nei sistemi di coltura all’esterno. Abbiamo sfruttato questo mutante con una marcata riduzione nel NPQ per verificare qual è l’impatto di questi meccanismi nella risposta di N.gaditana alla luce FL. Nel capitolo 5 quindi è stato messo a punto un esperimento in cui le colture sono state esposte ad una intensità luminosa di 150 μmol di fotoni m-2s-1, i quali nel controllo sono somministrati in modo costante, mentre nelle FL sono frutto di una combinazione di una bassa luce di base inframmezzata da flash di alta luce, dati con frequenze e durate diverse. Il risultato è un forte impatto delle FL sulla crescita del WT. Il difetto di crescita è proporzionale alla frequenza dei flash ed è più marcato alle frequenze maggiori. Tuttavia I48 non mostra una sensibilità maggiore a questo genere di trattamento rispetto al WT, perciò il NPQ non sembra essere coinvolto in modo significativo nella risposta di N.gaditana alla luce FL. Approfondite analisi sulla funzionalità dell’apparato fotosintetico hanno evidenziato che l’attività del PSII non è compromessa, bensì è il PSI ad essere pesantemente inattivato. Questo risultato suggerisce che i trasporti alternativi attorno al PSI, che in altri organismi fotosintetici sono attivati per evitare la sovraeccitazione del PSI, non sono così efficienti in N.gaditana. I primi 5 capitoli quindi hanno evidenziato il potenziale dell’ingegnerizzazione delle microalghe per trovare ceppi più produttivi rispetto al WT. Gli esperimento in FL in particolare non solo hanno aiutato a chiarire il ruolo del NPQ in questa microalga, ma tenendo conto che la FL è una condizione facilmente trovabile in sistemi industriali che utilizzano la luce del sole come fonte di energia, può fornire un punto di partenza per trovare nuovi targets di ingegnerizzazione per aumentare la produttività. Inizialmente ci siamo focalizzati su un approccio inserzionale, perché avrebbe dovuto facilitare l’identificazione dei geni responsabili del fenotipo. In questo caso la frequente presenza di inserzioni in tandem e l’accumulo di mutazioni puntiformi ha vanificato questo vantaggio, perciò strategie alternative sono in fase di ottimizzazione per il futuro. Nel capitolo 6 abbiamo invece testato l’approccio di mutagenesi chimica ed il protocollo di selezione, ottimizzati per N.gaditana, su un’altra specie: Scenedesmus obliquus, per valutare se questo metodo può essere applicato con successo a qualsiasi specie di interesse. Almeno un ceppo promettente è stato isolato (SOB17). In questo lavoro si è messo a punto anche un diverso sistema di valutazione delle produttività, attraverso un pannello messo in coltura in continuo. Infatti la geometria del pannello migliora la distribuzione della luce, rendendola più omogenea rispetto ad altri sistemi come la bottiglia. Inoltre la coltura in continuo fornisce una coltura stabile che ha una produzione continua di biomassa. Questa coltura ha avuto una buona stabilità nel tempo, arrivando ad essere, mantenuta per più di 30 settimane e SOB17 ha mostrato anche una maggiore produttività in una delle condizioni testate, anche se di nuovo come si era visto per N.gaditana, le condizioni di coltura sono fondamentali per ottenere il miglior risultato da ogni ceppo. In aggiunta all’Appendice 1 descritta prima, altre due appendici sono incluse nella tesi. Queste sono il risultato di due diverse collaborazioni in cui gli approcci sperimentali messi a punto in questa tesi sono stati applicati con fini diversi. In Appendice 2 è riportato il lavoro già pubblicato “Genetic Engineering of algae photosynthetic productivity using mathematical models” che descrive lo sviluppo di un modello matematico per valutare i fattori che influenzano la produttività delle microalghe nei fotobioreattori. Il modello è in grado di predire gli effetti di modifiche genetiche in un contesto di produzione industriale, fornendo uno strumento per identificare i genotipi più vantaggiosi in termini di produttività. In Appendice 3 è riportata una collaborazione con la Prof.ssa Angela Falciatore (Diatom Functional Genomics team, UPMC-Paris). In questo lavoro è stato adattato il protocollo di selezione ottimizzato per N.gaditana per trovare fenotipi fotosintetici in una collezione di mutanti di Phaeodactylum tricornutum, ottenuti per RNA interference così da modulare l’espressione di diversi fattori di trascrizione. Con questo approccio è stato possibile stabilire alcune promettenti correlazioni tra fenotipo e modulazione di una determinata classe di fattori di trascrizione, che ora saranno validati con un’analisi approfondita del fenotipo di questi mutanti e dei livelli di trascrizione in questi ceppi mutati.

Lactic acid bacteria (LAB), a group of generally recognized as safe (GRAS) organisms that metabolize sugars into primarily lactic acid, have traditionally been used for the fermentation and preservation of various foods and beverages. There is increasing interest in the genetic manipulation of LAB to improve existing characteristics or introduce novel, industrially pertinent phenotypes. However, because these bacteria have food-related applications, their genetic modification requires the use of food-grade genetic engineering tools. LAB plasmids, self-replicating extrachromosomal DNA molecules, can be used to derive food-grade cloning vectors. The rationale of this research was to develop a food-grade cloning vector using a lactobacilli cryptic plasmid and to investigate its cloning and expression properties. The main objectives were to (i) screen Lactobacillus spp. for plasmids, (ii) isolate and characterize a plasmid, and (iii) use the plasmid replicon to construct a cloning vector and express heterologous genes in various hosts. This is the first step in the development of a new family of food-grade cloning vectors for the genetic modification of lactobacilli.

The nodulin-23 gene is an abundantly transcribed soybean gene induced in nodules during symbiosis with Rhizobium. Sequencing of the cDNA and genomic clones revealed one intron within an open reading frame. A 24,275 dalton protein was predicted. The transcription of nodulin-23 gene occurs concomitantly with Lbc$ sb3$ and nodulin-24 genes. The 5$ sp prime$-regions of nodulin-23 and Lbc$ sb3$ genes were sequenced and compared with that of nodulin-24. Three potential cis-regulatory sequences were identified. The presence of trans-acting molecule(s), possibly regulating the expression of these genes, was tested for in vitro by preincubating nuclei from embryonic axes with nodule extract and assaying for gene activation. Nodulin-23, nodulin-24, and Lbc$ sb3$ genes were specifically activated and demonstrated similar kinetics. Several genes used as controls were not stimulated. A nodule factor(s) was shown to bind the 5$ sp prime$-region of nodulin-23 gene. The corresponding DNA regions from the other two coordinately expressed nodulin genes specifically competed for this binding, whereas other genes did not bind this factor at all.