Biobutanol represents a second generation biofuel, which can be producedfrom renewable resources by microorganisms. A Saccharomyces cerevisiae strainbearing the five butanol synthetic genes (hbd, adhe2, crt, ccr and ERG10) wasconstructed, where the hbd, adhe2, crt and ccr genes are derived from Clostridiumbeijerinckii, while ERG10 is a yeast gene. The genes were transformed individually onsingle cassettes, which integrated into specific chromosomal sites. The single integrantstrains were back‐crossed to create a strain bearing all five butanol synthetic genes. The butanol synthetic enzymes appeared to be highly expressed in the cytosol,however, very little butanol was obtained (< 10 ppm). Therefore, additional geneticmanipulations were made with a view to restoring any redox imbalance channellingthe carbon flux toward the butanol pathway. Deletion of the ADH1 gene in strains withthe butanol pathway improved production to ~250 ppm (203 mg/L) butanol. Furtherimprovement to 360 ppm (292 mg/L) was gained by overexpressing the ALD6 and ACS2genes, that are involved in synthesis of acetyl‐CoA; the precursor for butanolbiosynthesis. However, the replacement of ALD6 with ALD2, which produces NADHinstead of NADPH, didn’t improve butanol yields. In addition, no significantimprovement of butanol yield was obtained when dehydrogenase enzymes from theglycerol biosynthetic pathway were deleted. An initial assessment of the bestconditions for butanol production were semi‐anaerobic growth at 30°C in 2% glucosewith a starting OD600 of 0.1.In this project, another key question was addressed: does the sensitivity of cellsto short chain alcohols like butanol affect butanol production? Previous work in theAshe lab has identified specific point mutations in the translation initiation factor,eIF2B, which generate resistance or sensitive phenotypes to exogenously addedbutanol. Here a comparison of butanol production in sensitive and resistantbackgrounds showed that the butanol yield was 1.5‐2 fold higher in a butanol resistantstrain compared to the sensitive mutant. Generating a ‘super’ butanol resistant strainbearing a GCD2‐S131A mutation in eIF2B promoted a higher butanol yield per cell. However, another consequence of this mutation was reduced growth. So thecombination of these effects meant that the overall butanol concentration in mediawas similar to the control. Overall this work highlights that S. cerevisiae can producebutanol but that further optimisation both at the level of the strain and processengineering would be necessary before this would be of interest to the commercialsector.

Lepidopteran insects like Helicoverpa armigera, more commonly known as the cotton bollworm, are economically important pests of a wide variety of crops throughout the world. The Helicoverpa armigera stunt virus (HaSV), a tetravirus with a bipartite single-stranded positive-sense RNA genome, has great potential as a biological pesticide against H. armigera. The larger genomic strand of this virus (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71 kDa capsid protein precursor (p71). 240 copies of p71 assemble into a procapsid with the concomitant encapsidation of the viral RNA. This is followed by a complex maturation event that is characterized by the autoproteolytic cleavage of p71 into the 64 kDa capsid protein (P64) and a 7 kDa peptide (p7). The rearrangements that occur during maturation results in the formation of mature HaSV capsids that can thereupon deliver RNA to other susceptible host cells. The principal objective of the research described in this study was to demonstrate that this virus could be assembled in Saccharomyces cerevisiae. S. cerevisiae expression vectors were constructed for the production of p71. This protein was detected in cell lysates from two different strains of S. cerevisiae, both containing either chromosomal or episomal copies of an expression cassette for P71. A number of factors relating to the expression of P71 (e.g. strains used, expression loci and expression rate) and the preparation of protein extracts from S. cerevisiae (e.g. the presence of various protease inhibitors and salt concentrations) were examined to attain optimal levels of soluble p71. A small fraction of the optimized soluble p71 was shown to be in the form of virus-like particles (VLPs), with a yield of ≤10⁷ VLPs from a 1.5l culture of P71⁺ cells. These particles were exclusively in the procapsid form, had a similar buoyant density to that of wild-type HaSV and could undergo maturation when the pH was reduced to 5. S. cerevisiae vectors were constructed for the episomal expression of the HaSV genomic RNAs. These vectors directed the transcription of RNA1 and RNA2 transcripts, which had similar sizes to those of the HaSV genomic RNAs. Mature HaSV particles were purified from cells, transgenic for P71, RNA1 and RNA2, by way of two different virus purification protocols that were developed during this study. RT-PCR analyses on RNA-extracts from these particles demonstrated that RNA transcripts, which were produced in trans with p71, could be encapsidated by HaSV capsids in S. cerevisiae. A droplet-feed bioassay on H. armigera larvae demonstrated that the S. cerevisiae-derived HaSV particles caused impaired larval development. This response was correlated with the detection of HaSV RNA2 in RNA extractions from larvae that were used in this bioassay. The results that were generated through the course of this study, provided proof for the concept of the non-host production of infectious HaSV particles from S. cerevisiae. This work could serve as a foundation for future research on the development of an expression system for the large-scale production of this virus as a biopesticide.

Uga3p, a member of the zinc binuclear cluster transcription factor family, is required for [gamma]-aminobutyric acid-dependent transcription of the UGA genes in Saccharomyces cerevisiae. Crystallographic data of some of the protein-DNA complexes of this family reveal that members of this family bind to CGG triplets. A conserved 19-nucleotide activation element in certain UGA gene promoter regions contains a CCG-N4-CGG everted repeat, proposed to be the binding site of Uga3p, UAS[subscript GABA]. The spacer region (N4) between the CGG triplets has been suggested to be the specificity determinant for binding to UAS[subscript GABA]. The data available from the Saccharomyces genome database indicates that there are multiple repeats of -CCG-N4-CGG- regions within the genome. These transcription factors are involved in the activation of specific pathways and the question arises as to how their specificity of binding is determined. The aim of this study was to understand the binding characteristics of Uga3p to UAS[subscript GABA] and to determine the affinity and specificity of this interaction. In this study, full-length (tagged and untagged) and truncated (1-124 a.a.) Uga3p was produced in a heterologous expression system (E. coli). The interaction of Uga3p with UAS[subscript GABA] in Saccharomyces cerevisiae was characterized in terms of binding in vitro and the transcriptional activation of lacZ reporter genes in vivo. The Uga3p was capable of binding to these sites in vitro independent of exogenous GABA. Electrophoretic mobility shift assays (EMSA) of the full-length Uga3p with the wild type UAS[subscript GABA] sequences produced two distinct mobility complexes. The complexes formed in the EMSA of the full-length Uga3p were those specific to the interaction of the Uga3p to UAS[subscript GABA]. The truncated Uga3p(1-124 a.a.), which has the DNA-binding zinc cluster domain, the linker region and the putative coiled-coil domain was not functionally equivalent to the full-length protein with respect to binding in vitro because the EMSAs of the UAS[subscript GABA] with the truncated Uga3p produced indistinct complexes. EMSAs using mutant UAS[subscript GABA] sequences and heterologously-produced full-length Uga3p, demonstrated that UAS[subscript GABA] consists of two, independent Uga3p-binding sites. This work presents evidence that the two Uga3p molecules bound to UAS[subscript GABA] most likely interact with each other. Unlike other zinc cluster binding sites the Uga3p-binding site is an asymmetric site of 5’-SGCGGNWWT-3’ (S= G or C, W = A or T and N = no nucleotide or G or C). UAS[subscript GABA] is a palindrome containing the two asymmetric Uga3p-binding sites. The two-site consensus sequence required for the binding of Uga3p to the UAS[subscript GABA] is present upstream of UGA1 (region -387 to -370) and UGA4 (region -403 to -387). Furthermore, a single Uga3p-binding site was identified in the 5’ untranslated regions of UGA2 (region -219 to -211). GABA-dependent transcriptional activation by UAS[subscript GABA] in vivo could be directly correlated to a high affinity, specific interaction of two Uga3p molecules to this UAS. Binding with high affinity required the conserved sequences flanking the everted repeat. This study provided evidence that the binding pattern of Uga3p is novel compared to other zinc cluster motifs investigated, as the sequences flanking the everted repeat are important regions for recognition by Uga3p. The studies with the truncated Uga3p (1 –124 a.a.), also suggested that the regions C-terminal to the DNA-binding motif and putative coiled-coil area of this protein are important for Uga3p-specific interactions with UAS[subscript GABA]. Investigation of regions C-terminal to the zinc cluster, linker and putative coiledcoil revealed an eight-motif regulatory region similar to that in other zinc cluster proteins. This indicated that the regions C-terminal to these domains are important for the regulation and activity of these proteins. A putative seven repeat WD40-like motif was identified within this region. This putative domain has been speculated to be important for protein-protein interactions. Phosphorylation and dephosphorylation in other proteins of this class have been indicated to be important for the regulation of the activity of these proteins. The bioinformatic analysis of Uga3p revealed two possible cAMP/cGMP-dependent protein kinase phosphorylation sites, four putative protein kinase C phosphorylation motifs and four putative casein kinase II phosphorylation motifs. This study has contributed to the understanding of the nature of interactions between Uga3p and its specific UAS [subscript GABA] and how the regions flanking the everted repeat determine its specificity. The comparison of the nature of the binding of truncated and full-length Uga3p in vitro provided evidence for the role played by the full-length protein in determining this specific interaction. This evidence suggested that the in vitro binding evidence for other proteins of this family, using truncated peptides that carry the DNA-binding domain, might not reflect the true nature of interactions between the proteins of this class and their specific UASs in vivo.

Submitted by Fernanda Rodrigues de Lima (fernanda.rlima@ufpe.br) on 2018-10-09T21:10:29Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Dayvison Soares Ferreira.pdf: 1198693 bytes, checksum: 380d0a706f7aa1cc0632a0069ea59a3a (MD5) / Approved for entry into archive by Alice Araujo (alice.caraujo@ufpe.br) on 2018-11-21T19:33:23Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Dayvison Soares Ferreira.pdf: 1198693 bytes, checksum: 380d0a706f7aa1cc0632a0069ea59a3a (MD5) / Made available in DSpace on 2018-11-21T19:33:23Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Dayvison Soares Ferreira.pdf: 1198693 bytes, checksum: 380d0a706f7aa1cc0632a0069ea59a3a (MD5) Previous issue date: 2015-03-31 / CAPES / A fermentação alcóolica é um processo amplamente usado no setor sucroalcooleiro brasileiro que usa a cana de açúcar como a principal matéria prima e a levedura da espécie Saccharomyces cerevisiae como agente fermentativo. Muito se conhece sobre o funcionamento da fermentação e as diversas variáveis físicas, químicas e biológicas a que ela está sujeita. No entanto, dentre essas variáveis, o papel da suplementação mineral ainda não foi bem esclarecido, fato que se deve as variações na composição química das matérias-primas decorrentes do tipo de solo, variedade da cana de açúcar e suas condições de cultivo. Sabe-se que o uso inadequado da suplementação mineral pode diminuir o rendimento fermentativo, além de causar toxicidade celular nas células de levedura do processo. Por isso, o objetivo desse estudo foi avaliar o efeito do magnésio e do cobre no metabolismo de S. cerevisiae, íons esses comumente usados nas suplementações minerais. As amostras de caldo de cana e melaço utilizadas no estudo foram coletadas em destilarias localizadas na Paraíba e em Pernambuco, respectivamente. Estes meios foram fracionados em duas amostras, uma para a análise da composição mineral de Cu⁺⁺ e Mg⁺⁺ no Instituto de Tecnologia de Pernambuco e a outra para a análise do crescimento celular e realização dos ensaios fermentativos para avaliação dos metabólitos produzidos, como glicerol, ácidos e álcool a partir dos açúcares presentes nos meios. Todos os ensaios foram realizados com a linhagem industrial JP1 da S.cerevisiae por ela ser utilizada com maior frequência em fermentações industriais da região Nordeste. Após os ensaios, constatou-se que o magnésio tem um papel importante no direcionamento metabólico de S.cerevisiae, estimulando a rota fermentativa da levedura e inibindo a produção de biomassa pela célula. Ao se analisar o ensaio fermentativo, verificou-se que os nas amostras de caldo de cana suplementados apresentaram um maior rendimento fermentativo, porém nas amostras de melaço suplementadas, houve uma redução no rendimento fermentativo. Quanto à toxicidade mineral para a levedura, nenhum dos dois minerais testados afetou a viabilidade e o brotamento celular, estas permanecendo com valores > 95% e >5%, respectivamente 20 horas após o término da fermentação. Ao compararmos o efeito do magnésio em diferentes meios de cultura e com diferentes minerais (cálcio e manganês) além do cobre, concluímos que a concentração do magnésio possui uma grande influência no direcionamento metabólico de S. cerevisiae, mais do que a própria concentração mineral no meio. / The alcoholic fermentation is a widely used process in the Brazilian sugar and ethanol industry that uses sugarcane as the main raw material and the yeast Saccharomyces cerevisiae species as fermentation agent. Much is known about the functioning of fermentation and the several variables physical, chemical and biological to which it is subject. However, among these variables, the role of mineral supplementation is not yet well understood, a fact that is due to the variations in the chemical composition of raw materials resulting from soil type, variety of sugarcane and your cultivation. It is known that misuse of mineral supplementation can reduce the fermentative yield and cause cell toxicity in the process the yeast cells. Therefore, the aim of this study was to evaluate the effect of magnesium and copper in the metabolism of S. cerevisiae, these ions commonly used in the mineral supplementation. Samples of sugarcane juice and molasses used in the study were collected in distilleries located in Paraíba and Pernambuco, respectively. These medium were fractionated into two samples, one for analyzing the mineral Cu ++ and Mg ++ composition in Instituto de Tecnologia de Pernambuco and another for analysis of cell growth and carrying out fermentation test for evaluating the produced metabolites such as glycerol, acids and alcohol from the sugars present in the medium. All assays were performed with the JP1 industrial strain of S. cerevisiae because it is used more frequently in industrial fermentations in the Northeast. After the tests, it was found that magnesium has an important role in the metabolic targeting of S. cerevisiae, stimulating the fermentation route and inhibiting yeast biomass production by the cell. When analyzing the fermentation test, it was found that the sugarcane juice supplemented samples exhibited an increased fermentation yield, but in molasses supplemented samples, there was a reduction in fermentation yield. As the mineral toxicity to yeast, neither tested mineral affected cell viability and budding, these remaining values with> 95% and> 5%, respectively, 20 hours after fermentation. Comparing the effect of Mg in different culture media with different mineral (calcium and manganese) in addition to copper, we conclude that the concentration magnesium has a great influence on the metabolic S. cerevisiae targeting more than the actual mineral concentration medium.

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