Generation and phenotypic analyses of stzA gene deletion strains of Aspergillus nidulans implies that stzA is allelic to sltA, with the encoded transcription factor regulating tolerance to cations, DNA-damaging agents and high arginine concentrations. The similar severe sensitivity of a sltA1 mutant (GO281) and stzA deletion mutants to these stresses indicated that the premature termination codon in sltA1 represents a total loss-of-function mutation. It was also verified that StzA has no regulatory role in the utilisation of carbon sources. Findings were supported by phenotypic analyses of recombinant progeny resulting from sexual crosses between sltA1 and sltA+ strains. Bioinformatic analysis of genes involved in the osmotic stress response revealed that their promoters were significantly enriched for StzA binding site motifs compared to those of the control group, indicating that StzA may regulate many of these genes that comprise the High Osmolarity Glycerol (HOG) pathway. Although this pathway is activated by fludioxonil, stzA deletants and stzA+ strains showed similar sensitivities to this fungicide. Phenotypic analyses indicate that StzA does not regulate tolerance to sources of oxidative stress, non-ionic osmotic stress or components of the Cell Wall Integrity (CWI) pathway. A. nidulans StzA appears to have no role in cellulase or xylanase expression as revealed by the results of a dinitrosalicylic acid (DNS) assay. Trichoderma reesei ace1 deletant and wild-type strains showed similar sensitivities to cations, DNA-damaging agents, arginine, neomycin, acidic and alkaline pH. These results confirm that A. nidulans StzA and T. reesei Ace1 regulate the distinct phenotypes of abiotic stress tolerance and cellulase and xylanase expression, respectively, despite these two proteins sharing 58% overall amino acid similarity. All twenty-nine StzA orthologues identified are restricted to filamentous ascomycetes of the Pezizomycotina subphylum and may therefore represent specific and novel antifungal drug targets. The C-termini of StzA proteins are highly variable in both length and sequence, with no apparent conservations in amino acids or predicted secondary structure. This region is considered most likely to influence the divergent functions of StzA proteins. Conservations of individual residues in the N-termini correspond to conserved secondary structure (alpha helices) among StzA proteins, implying shared functions for StzA proteins in this region. Regulators of two major nitrogen metabolic pathways (CpcA and AreA) may regulate stzA expression. Statistically significant putative CpcA binding sites were positionally conserved in 26 out of 29 stzA orthologue promoters, indicating an interaction between stzA and CpcA, a transcription factor that mediates the cross pathway control of amino acid biosynthesis. REALALE sequences, likely to be of retrotransposon origin, containing putative overlapping binding sites for StzA and AreA, were found in eleven stzA promoters of the Eurotiomycetes class, indicating an interaction between stzA and the global nitrogen metabolite repressor AreA. Intriguingly, REALALE-containing promoters identified across the genome of A. nidulans were significantly enriched for StzA binding site motifs when compared to a control group of genes. Hence, REALALE may have regulatory significance that extends to other A. nidulans genes.

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2018-05-25T11:53:11Z No. of bitstreams: 2 Indianara Kawana Bueno.pdf: 2050489 bytes, checksum: 3865a86596759f1ff761267b04d8a615 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-25T11:53:11Z (GMT). No. of bitstreams: 2 Indianara Kawana Bueno.pdf: 2050489 bytes, checksum: 3865a86596759f1ff761267b04d8a615 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-09 / The research for renewable energy sources became even more essential due the imminent depletion of the fossil fuel sources. In this context Brazil has a prominent position on the world stage, since it has already used ethanol from sugar cane for some decades. The second generation ethanol (2G) is produced from the lignocellulosic biomass of the vegetable, which is composed by cellulose, hemicellulose and lignin. The hydrolysis of these compounds requires a specific and high cost enzymatic cocktail. On this scenario, the Trichoderma reesei fungus gains spotlight, since it is one the microorganisms with the highest potential to produce hydroliytic enzymes. Therefore, the attempt to increase the cellulases production of this fungus is an important for the production of biofuels more attractive to the market. The aim of this work is to confirm the deletion of the sequence which codifies the zinc finger motif of the transcription factor ACE1 for cellulose repression from the T. reesei RUT-C30 strain and to characterize the enzymatic production of these mutant strains named T. reesei RUT-C30Δzface1. The enzymatic quantification was carried using the substrates carboxymethyl cellulose, microcrystalline cellulose and Whatman paper filter. The deletion confirmation occurred by the absence of the amplification gene ace1 on the mutants and the amplification of a 429 pb fragment of the RUT-C30 parental strain when the same primers and PCR conditions where used. These results suggest that the deletion of the zinc finger motif of the from ACE1 transcription factor is a prominent way to achieve an economically viable production of bioethanol. / Com a depleção eminente das fontes de combustíveis fósseis, torna-se cada vez mais imprescindível a busca por fontes renováveis de energia. Neste âmbito, o Brasil tem destaque no cenário mundial, pois já utiliza o etanol a partir da cana-de-açúcar há algumas décadas. O etanol de segunda geração (2G) é produzido a partir da massa lignocelulolítica do vegetal, que é composta de celulose, hemicelulose e lignina. A hidrólise desses compostos necessita de um coquetel enzimático específico e de alto custo. Neste cenário, o fungo Trichoderma reesei ganha destaque, pois é um dos microrganismos com maior potencial para produção de enzimas hidrolíticas. Desta forma, as tentativas de aumentar a produção de celulases desse fungo, torna a produção do bioetanol uma alternativa mais atrativa ao mercado. Este trabalho teve como objetivos confirmar a deleção da sequência que codifica o dedo de zinco do fator de transcrição do repressor de celulase ACE1 da linhagem T. reesei RUT-C30 e caracterizar a produção enzimática dessas linhagens mutantes denominadas T. reesei RUT-C30Δzface1. A confirmação de deleção ocorreu pela ausência de amplificação do gene ace1 nos mutantes e amplificação de um fragmento de 479 pb na linhagem parental RUT-C30, quando utilizados os mesmos primers e condições de reação de PCR. A dosagem enzimática com os substratos carboximetilcelulose (CMC), celulose microcristalina (Avicel®) e papel de filtro Whatman (PF), mostraram que o RUT-C30Δzface1 tem a atividade celulolítica aumentada em até 3,2 vezes em Avicel e 2,1 vezes em CMC e PF em comparação à linhagem parental RUT-C30. Em 24 horas de hidrólise os mutantes apresentaram liberação de açúcar 1,4 vezes maior em relação ao RUT-C30. Estes resultados sugerem que a deleção parcial do fator de transcrição ACE1 é um proeminente caminho para a conquista de uma produção de bioetanol economicamente viável.

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