RESUMO
Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (T(opt) 95-100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (T(opt) 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas-liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.
Assuntos
Dióxido de Carbono/metabolismo , Ácido Láctico/análogos & derivados , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Reatores Biológicos/microbiologia , Perfilação da Expressão Gênica , Temperatura Alta , Ácido Láctico/metabolismo , Maltose/metabolismo , Pyrococcus furiosus/efeitos da radiação , Sulfolobaceae/genéticaRESUMO
The remarkable survival of the hyperthermophilic archaeon Pyrococcus furiosus to ionizing radiation was previously demonstrated. Using a time course study and whole-genome microarray analyses of mRNA transcript levels, the genes and regulatory pathways involved in the repair of lesions produced by ionizing irradiation (oxidative damage and DNA strand breaks) in P. furiosus were investigated. Data analyses showed that radA, encoding the archaeal homolog of the RecA/Rad51 recombinase, was moderately up regulated by irradiation and that a putative DNA-repair gene cluster was specifically induced by exposure to ionizing radiation. This novel repair system appears to be unique to thermophilic archaea and bacteria and is suspected to be involved in translesion synthesis. Genes that encode for a putative Dps-like iron-chelating protein and two membrane-bound oxidoreductases were differentially expressed following gamma irradiation, potentially in response to oxidative stress. Surprisingly, the many systems involved in oxygen detoxification and redox homeostasis appeared to be constitutively expressed. Finally, we identified several transcriptional regulators and protein kinases highly regulated in response to gamma irradiation.
Assuntos
Proteínas Arqueais/genética , Raios gama , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica em Archaea/efeitos da radiação , Análise de Sequência com Séries de Oligonucleotídeos , Pyrococcus furiosus/efeitos da radiação , RNA Arqueal/metabolismo , RNA Mensageiro/metabolismo , Proteínas Arqueais/metabolismo , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Replicação do DNA/genética , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta à Radiação , Proteínas de Ligação ao Ferro/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/efeitos da radiação , Oxirredutases/metabolismo , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Temperatura , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.