ABSTRACT: Experimental evolution of a hyperthermophilic archaeon on carbon monoxide enhanced hydrogen productivity through novel mechanisms associated with genome, transcriptome and epigenome changes
Project description:Hyperthermophilic archeaon, Thermococcus onnurineus NA1 has known as a strict anaerobe. To date, a few of studies have been reported that strict anaerobe can grow using oxygen. However, the research of the growth enhancement of strict anaerobic archaeon belonging to the order of Thermococcales using the oxygen, in which has never been reported so far. In this study, we showed that the growth of T. onnurineus NA1 strain increased under various oxygen concentrations and we observed that oxygen was decreased in the headspace during the growth of cell. Genome-wide transcriptomic analysis was carried out to evaluate alterations in gene expression induced by O2 and to explain the physiological effects of oxidative stress on the growth of T. onnurineus NA1.
Project description:A hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on a variety of CO-containing feed stocks such as by-product gas generated from steel-mill process. In this study we applied a long-term adaptive evolution to enhance H2 productivity. Through serial transfer of cell cultures with carbon monoxide (CO) as an energy source, we observed physiological changes in cell density, CO consumption rate and H2 production rate. To understand the underlying mechanism for the changes, we performed systems analysis of genomic, transcriptomic and epigenomic data. Genomic analysis of an evolved strain, designated as 156T, revealed that single or multiple bases were substituted, deleted or inserted in the sequence of the parental strain. A single point mutation in a putative transcriptional regulator (TON_1525) seemed to play a pivotal role in changing cellular phenotypes by increasing the expression level of genes of a CO dehydrogenase-hydrogenase gene cluster. Additionally, a mutation in an aromatic amino acid permease (TON_0820) contributed to increasing cell growth. Transcriptomic analysis revealed that genes belonging to the categories of transcription, translation and energy metabolism in archaeal Clusters of Orthologous Genes (arCOGs) were significantly changed. In particular, genes involved in energy conservation via CO oxidation were highly upregulated. Epigenomic analysis suggested that methylation change might be a way of gene regulation in a hyperthermophilic archaeon. The evolved 156T strain showed highly enhanced hydrogen productivity with CO at high flow rates of 800 ml min-1 and above, indicating the adaptation rendered the strain less sensitive to high CO. The 156T strain was demonstrated to be appropriate for H2 production by using synthetic gas obtained by coal gasification. This study is the first example to show that evolutionary engineering is very effective in enhancing H2 productivity of a hyperthermophilic archaeon on CO
Project description:Expression analysis of Thermococcus onnurineus NA1 KCTC10859 and an Frh-deficient mutant, Frh overexpression mutant and TON_0282 deletion mutant in a T. onnurineus NA1 strain.
Project description:Lon is an oligomeric ATP-dependent protease that degrades defective or denatured proteins as well as some folded proteins for the control of cellular protein quality and metabolism. Lon from Thermococcus onnurineus NA1 was purified and crystallized at 295 K. A 2.0 A resolution data set was collected using synchrotron radiation. The crystals belonged to space group P6(3), with unit-cell parameters a = 121.45, b = 121.45, c = 195.24 A. Assuming the presence of two monomers in the asymmetric unit, the solvent content was estimated to be about 60.7%.
Project description:Two hypothetical genes were functionally verified to be a pyrophosphatase and a PAP phosphatase in Thermococcus onnurineus NA1. This is the first report of the pyrophosphatases and the PAP phosphatases being organized in the gene clusters of the sulfate activation system only in T. onnurineus NA1 and "Pyrococcus abyssi."
Project description:The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H? when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of T. onnurineus NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H?-evolving substrate culture conditions. Using label-free nano-UPLC-MSE-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins) was found to be significantly up-regulated (?1.5-fold) under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe-S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H?-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments.
Project description:Thermococcus onnurineus NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S0) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5'-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from Thermococcus onnurineus NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures. The catalytic intermediate structures showed the dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring. The ToCDS structures were compared with bacterial CDS structures, which will help us to understand the role and catalytic mechanism of ToCDS in the archaeon Thermococcus onnurineus NA1.
