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Doctoral Thesis
DOI
10.11606/T.87.2008.tde-06082009-111247
Document
Author
Full name
Juliana Carvalho de Arruda Caulkins
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2008
Supervisor
Committee
Rodrigues, Maria Filomena de Andrade (President)
Alterthum, Flavio
Antonio, Regina Vasconcellos
Gomez, José Gregorio Cabrera
Maiorano, Alfredo Eduardo
Title in Portuguese
Identificação de genes envolvidos na síntese de polihidroxialcanoatos em Burkholderia cepacia linhagem IPT64.
Keywords in Portuguese
b-cetotiolase
Burkholderia cepacia
Biotecnologia
Genética bacteriana
PHA- Sintase
PHB
Polihidroxialcanoatos
Polímeros biodegradáveis
Abstract in Portuguese
Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado.
Title in English
Identification of genes involved in the synthesis of polyhydroxyalkanoates on Burkholderia cepacia strain IPT64.
Keywords in English
b-cetotiolase
Burkholderia cepacia
Biodegradable polymers
Biotechnology
Genetics bacterial
PHA-synthase
PHB
Polyhydroxyalkanoates
Abstract in English
The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.
 
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Publishing Date
2009-08-26
 
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