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Master's Dissertation
DOI
10.11606/D.87.2011.tde-15092011-142026
Document
Author
Full name
Keina Poliana Pivarro Dalmolin
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2011
Supervisor
Committee
Vicente, Elisabete Jose (President)
Rivera, Irma Nelly Gutierrez
Rubio, Ileana Gabriela Sanchez de
Title in Portuguese
Clonagem de um alelo do gene SPT15 em Saccharomyces cerevisiae para aumento da produção de etanol.
Keywords in Portuguese
Saccharomyces
Clonagem
Etanol
Genes
Genética microbiana
Glicose
Abstract in Portuguese
Alper e colaboradores demonstraram que a linhagem BY4741 recombinante portadora de cópias adicionais de um alelo SPT15 mutagenizado em três diferentes posições (spt15-300) utiliza mais rapidamente a glicose e aumenta a produção de etanol. Neste trabalho, foi realizada a clonagem deste alelo, aqui chamado spt15*. Inicialmente o DNA genômico da linhagem S. cerevisiae S288C foi utilizado como molde para amplificação por SOEing-PCR. O alelo spt15* foi clonado no plasmídeo pGEMT-Easy e, em seguida, introduzido no plasmídeo epissomal pMA91. Após construções moleculares, foi obtido o fragmento de DNA dpPGKspt15*tPGKd, empregado na transformação genética, da linhagem S. cerevisiae YPH252, por d-integração. Os clones recombinantes YHP252/pMA91spt15* e YHP252/dpPGKspt15*tPGKd consomem mais eficientemente a glicose e aumentam a produção de etanol. O seqüenciamento do alelo SPT15 da levedura industrial PE-2 revelou 100% de identidade com o alelo das linhagens BY4741 e S288C, criando ótimas perspectivas para trabalhos futuros.
Title in English
Cloning of an SPT15 allele gene in Saccharomyces cerevisiae for the increasing of ethanol production.
Keywords in English
Saccharomyces
Cloning
Ethanol
Genes
Glucose
Microbial genetics
Abstract in English
Alper and colleagues demonstrated that the yeast S. cerevisiae BY4741 recombinant strain carrying additional copies of a SPT15 allele mutagenized in three different positions (spt15-300) uses glucose more speedily and produces more ethanol. In this work, this allele, here called spt15*, was cloned. The genomic DNA of the S. cerevisiae S288C strain was used for amplification through SOEing-PCR. The spt15* allele was cloned in the plasmid pGEMT-Easy and introduced in the episomal plasmid pMA91. After molecular constructions the DNA dpPGKspt15*tPGKd fragment was obtained to be employed in the genetic transformation of laboratory S. cerevisiae strains using d-integration. Both recombinant clones YHP252/pMA91spt15* and YHP252/dpPGKspt15*tPGKd consume glucose more speedily and produce more ethanol. The sequencing of the SPT15 allele of the industrial yeast PE-2 revealed 100% identity with BY4741 and S288C alleles, creating huge perspectives for future works.
 
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Publishing Date
2011-09-22
 
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