Prospecção de genes com interesse biotecnológico
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2011-06-21
Autores
Schuch, Viviane [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Neste trabalho foi realizada a prospecção de novos genes de interesse biotecnológico em bibliotecas metagenômicas e em linhagens de Burkholderia através da técnica de reação em cadeia da polimerase. Nas bibliotecas metagenômicas, foi possível identificar genes que codificam álcool desidrogenase, oxirredutase, acil-CoA desidrogenase, luciferase, transportadores de membrana, thiolase, aminoglicosídeo fosfotransferase, desidrogenase de cadeia curta, proteínas repressoras TetR, enoil-CoA hidratase/isomerase, entre outras. Nas treze linhagens de Burkholderia testadas, seis apresentaram amplificação positiva para o gene de xilose isomerase. Estes genes foram completamente sequenciados e as sequências foram utilizadas em análises computacionais, que permitiram estabelecer a identidade entre as sequências e a dedução da função das proteínas baseado em similaridades. Foi realizada a medida do índice de adaptação de códons, com a finalidade de se encontrar um hospedeiro onde a expressão seja maximizada, baseando-se na presença de códons preferenciais e raros. Está análise mostrou que os genes encontrados possuem boas possibilidades de expressão em Escherichia coli, mas que podem apresentar uma taxa de expressão ineficiente em Saccharomyces cerevisiae. Foi realizado um teste de complementação gênica utilizando um dos genes descobertos e uma linhagem de Escherichia coli que possui o gene de xilose isomerase nocauteado. Os transformantes foram capazes de crescer em meio cuja única fonte de carbono era o açúcar xilose, mostrando que o gene é funcional. Estes genes serão utilizados futuramente em ensaios de expressão para caracterização da enzima
In this work was carried out prospection for new genes of biotechnological interest in metagenomic libraries and strains of Burkholderia through the technique of polymerase chain reaction. In metagenomics libraries, we could identify genes encoding alcohol dehydrogenase, oxidoreductase, acyl-CoA dehydrogenase, luciferase, membrane transporters, thiolase, aminoglycoside phosphotransferase, short-chain dehydrogenase, TetR repressor protein, enoyl-CoA hydratase/isomerase, among others. Of the 13 strains of Burkholderia tested, 6 showed positive amplification for the xylose isomerase gene. The genes were completely sequenced and the sequences were used in computational analysis, which allowed to establish the identity between the sequences and deducing protein function based on similarities. We evaluated the codon adaptation index, with the aim of finding a host in which the expression is maximized, based on the presence of preferred codons and rare. This analysis showed that the genes found have good possibilities of expression in Escherichia coli, but that may have an inefficient rate of expression in Saccharomyces cerevisiae. We conducted a genetic complementation test using one of the discovered genes and one strain of Escherichia coli that has the xylose isomerase gene knocked out. Transformants were able to grow in a medium whose sole source of carbon was the sugar xylose, showing that the gene is functional. These genes will be used in expression assays for characterization of new enzymes
In this work was carried out prospection for new genes of biotechnological interest in metagenomic libraries and strains of Burkholderia through the technique of polymerase chain reaction. In metagenomics libraries, we could identify genes encoding alcohol dehydrogenase, oxidoreductase, acyl-CoA dehydrogenase, luciferase, membrane transporters, thiolase, aminoglycoside phosphotransferase, short-chain dehydrogenase, TetR repressor protein, enoyl-CoA hydratase/isomerase, among others. Of the 13 strains of Burkholderia tested, 6 showed positive amplification for the xylose isomerase gene. The genes were completely sequenced and the sequences were used in computational analysis, which allowed to establish the identity between the sequences and deducing protein function based on similarities. We evaluated the codon adaptation index, with the aim of finding a host in which the expression is maximized, based on the presence of preferred codons and rare. This analysis showed that the genes found have good possibilities of expression in Escherichia coli, but that may have an inefficient rate of expression in Saccharomyces cerevisiae. We conducted a genetic complementation test using one of the discovered genes and one strain of Escherichia coli that has the xylose isomerase gene knocked out. Transformants were able to grow in a medium whose sole source of carbon was the sugar xylose, showing that the gene is functional. These genes will be used in expression assays for characterization of new enzymes
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Palavras-chave
Metagenome, Biocombustíveis, Xarope de milho, Metagenôma, Xilose isomerase
Como citar
SCHUCH, Viviane. Prospecção de genes com interesse biotecnológico. 2011. v, 80 f. Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2011.