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Perspectivas sobre polihidroxialcanoatos em microrganismos extremófilos: busca de genes relacionados a PHA e acúmulo de PHA em microrganismos halófilos recuperados de uma salina interior
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Abstract(s)
O uso de plásticos derivados de petróleo é uma problemática global para o meio ambiente, com esforços crescentes para mitigar seus efeitos. Estudos sobre bioplásticos, como os polihidroxialcanoatos (PHA), têm ganhado destaque. Estes poliésteres são similares aos plásticos convencionais, todavia são biocompatíveis, biodegradáveis e renováveis. Microrganismos halófilos dentro dos domínios Archaea e Bacteria já foram identificados como sintetizadores destes biopolímeros e enzimas participantes de vias metabólicas de PHA também. O presente trabalho teve como objetivo identificar em 7 estirpes de haloarquéias isoladas de uma amostra de um cristalizador da Salina de Rio Maior, Portugal, o acúmulo de PHA e a busca por genes relacionados ao metabolismo de PHA a partir do metagenoma da mesma amostra. A potencialização do acúmulo de PHA foi testada a partir de glicose ou glicerol, sendo esta detectada a partir de glicose. A detecção de grânulos de PHA nos isolados próximos de Halobacterium salinarum e uma nova espécie a ser descrita, "Halorubrum miltondacostae", foi possível através de micrografias de TEM e da coloração por Sudan Black B. A detecção de PHB nas estirpes de Haloarcula marismortui e "Halorubrum miltondacostae" foi confirmada a partir do ensaio do ácido crotónico. A extração de PHA foi realizada a partir de 6 metodologias distintas, destacando-se a extração de PHB utilizando SDS 0.1% ou água destilada como as mais eficazes. A detecção dos genes foi realizada a partir da busca destes no metagenoma e a comparação contra o Genbank corroborou a identificação da função dos genes. Foram detectados genes associados a vias metabólicas em representantes dos domínios Bacteria e Archaea. Genes codificantes para PhaA, PhaB, PhaC, PGAPs e de proteínas relacionadas a mobilização de PHA (PhaJ e PhaZ) foram detectados em Gammaproteobacteria e Rhodobacteraceae. Esses resultados destacam o potencial dos halófilos como produtores de PHA e a diversidade genética envolvida nesse processo.
The use of petroleum-derived plastics is a global environmental issue, with increasing efforts to mitigate its effects. Studies on bioplastics, such as polyhydroxyalkanoates (PHA), have gained prominence. These polyesters are similar to conventional plastics; however, they are biocompatible, biodegradable, and renewable. Halophilic microorganisms within the Archaea and Bacteria domains have been identified as synthesizers of these biopolymers, along with enzymes participating in PHA metabolic pathways. The current study aimed to detect PHA accumulation in seven haloarchaeal strains isolated from a crystallizer sample obtained from the Rio Maior Saltern, Portugal, and to investigate genes associated with PHA metabolism in the metagenome of the same sample. Enhancement of PHA accumulation was tested using glucose or glycerol, with glucose being detected as the preferred carbon source. Detection of PHA granules in isolates closely related to Halobacterium salinarum and a new species to be described, "Halorubrum miltondacostae," was possible through TEM micrographs and Sudan Black B staining. PHB detection in Haloarcula marismortui and "Halorubrum miltondacostae" strains was confirmed using the crotonic acid assay. The PHA extraction was performed using six different methodologies and the SDS 0.1% or distilled water extraction being the most effective for PHB extraction. Gene detection was conducted by searching for them in the metagenome, and comparison against GenBank corroborated gene function identification. Genes associated with metabolic pathways in representatives of Bacteria and Archaea domains were detected. Genes encoding PhaA, PhaB, PhaC, PGAPs, and proteins related to PHA mobilization (PhaJ and PhaZ) were detected in Gammaproteobacteria and Rhodobacteraceae. These results highlight the potential of halophiles as PHA producers, and the genetic diversity involved in this process.
The use of petroleum-derived plastics is a global environmental issue, with increasing efforts to mitigate its effects. Studies on bioplastics, such as polyhydroxyalkanoates (PHA), have gained prominence. These polyesters are similar to conventional plastics; however, they are biocompatible, biodegradable, and renewable. Halophilic microorganisms within the Archaea and Bacteria domains have been identified as synthesizers of these biopolymers, along with enzymes participating in PHA metabolic pathways. The current study aimed to detect PHA accumulation in seven haloarchaeal strains isolated from a crystallizer sample obtained from the Rio Maior Saltern, Portugal, and to investigate genes associated with PHA metabolism in the metagenome of the same sample. Enhancement of PHA accumulation was tested using glucose or glycerol, with glucose being detected as the preferred carbon source. Detection of PHA granules in isolates closely related to Halobacterium salinarum and a new species to be described, "Halorubrum miltondacostae," was possible through TEM micrographs and Sudan Black B staining. PHB detection in Haloarcula marismortui and "Halorubrum miltondacostae" strains was confirmed using the crotonic acid assay. The PHA extraction was performed using six different methodologies and the SDS 0.1% or distilled water extraction being the most effective for PHB extraction. Gene detection was conducted by searching for them in the metagenome, and comparison against GenBank corroborated gene function identification. Genes associated with metabolic pathways in representatives of Bacteria and Archaea domains were detected. Genes encoding PhaA, PhaB, PhaC, PGAPs, and proteins related to PHA mobilization (PhaJ and PhaZ) were detected in Gammaproteobacteria and Rhodobacteraceae. These results highlight the potential of halophiles as PHA producers, and the genetic diversity involved in this process.
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Keywords
Polihidroxialcanoatos (phas) Extremófilos Archaea Metagenoma Biossíntese Proteínas associadas aos grânulos (pgaps)