scholarly journals Metabolism Dealing with Thermal Degradation of NAD+ in the Hyperthermophilic Archaeon Thermococcus kodakarensis

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Shin-ichi Hachisuka ◽  
Takaaki Sato ◽  
Haruyuki Atomi
Keyword(s):  
Thermal Degradation ◽  
Mutant Strain ◽  
Physiological Role ◽  
Cell Extract ◽  
Host Strain ◽  
Enzymatic Oxidation ◽  
Oxidation Reactions ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis

ABSTRACT NAD+ is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD+ is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD+ concentrations and possibly remove and/or reuse undesirable degradation products of NAD+. Here we confirmed that at 85°C, thermal degradation of NAD+ results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD+ in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD+ breakdown. IMPORTANCE Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD+ is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal degradation to ADP-ribose and nicotinamide. Here we show that an ADP-ribose pyrophosphatase homolog from the hyperthermophilic archaeon Thermococcus kodakarensis converts the detrimental ADP-ribose to ribose 5-phosphate and AMP, compounds that can be directed to central carbon metabolism. This physiological role for ADP-ribose pyrophosphatases might be universal in hyperthermophiles, as their homologs are widely distributed among both hyperthermophilic bacteria and archaea.

scholarly journals Distinct Physiological Roles of the Three Ferredoxins Encoded in the Hyperthermophilic Archaeon Thermococcus kodakarensis

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Brett W. Burkhart ◽  
Hallie P. Febvre ◽  
Thomas J. Santangelo
Keyword(s):  
Electron Flux ◽  
Electron Flow ◽  
Physiological Role ◽  
High Energy ◽  
Electron Acceptors ◽  
Content Type ◽  
Maximal Energy ◽  
Thermococcus Kodakarensis ◽  
Small Proteins ◽  
Energy Gains

ABSTRACT Control of electron flux is critical in both natural and bioengineered systems to maximize energy gains. Both small molecules and proteins shuttle high-energy, low-potential electrons liberated during catabolism through diverse metabolic landscapes. Ferredoxin (Fd) proteins—an abundant class of Fe-S-containing small proteins—are essential in many species for energy conservation and ATP production strategies. It remains difficult to model electron flow through complicated metabolisms and in systems in which multiple Fd proteins are present. The overlap of activity and/or limitations of electron flux through each Fd can limit physiology and metabolic engineering strategies. Here we establish the interplay, reactivity, and physiological role(s) of the three ferredoxin proteins in the model hyperthermophile Thermococcus kodakarensis. We demonstrate that the three loci encoding known Fds are subject to distinct regulatory mechanisms and that specific Fds are utilized to shuttle electrons to separate respiratory and energy production complexes during different physiological states. The results obtained argue that unique physiological roles have been established for each Fd and that continued use of T. kodakarensis and related hydrogen-evolving species as bioengineering platforms must account for the distinct Fd partnerships that limit flux to desired electron acceptors. Extrapolating our results more broadly, the retention of multiple Fd isoforms in most species argues that specialized Fd partnerships are likely to influence electron flux throughout biology. IMPORTANCE High-energy electrons liberated during catabolic processes can be exploited for energy-conserving mechanisms. Maximal energy gains demand these valuable electrons be accurately shuttled from electron donor to appropriate electron acceptor. Proteinaceous electron carriers such as ferredoxins offer opportunities to exploit specific ferredoxin partnerships to ensure that electron flux to critical physiological pathways is aligned with maximal energy gains. Most species encode many ferredoxin isoforms, but very little is known about the role of individual ferredoxins in most systems. Our results detail that ferredoxin isoforms make largely unique and distinct protein interactions in vivo and that flux through one ferredoxin often cannot be recovered by flux through a different ferredoxin isoform. The results obtained more broadly suggest that ferredoxin isoforms throughout biological life have evolved not as generic electron shuttles, but rather serve as selective couriers of valuable low-potential electrons from select electron donors to desirable electron acceptors.

scholarly journals Identification of Dephospho-Coenzyme A (Dephospho-CoA) Kinase in Thermococcus kodakarensis and Elucidation of the Entire CoA Biosynthesis Pathway in Archaea

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Takahiro Shimosaka ◽  
Kira S. Makarova ◽  
Eugene V. Koonin ◽  
Haruyuki Atomi
Keyword(s):  
Metabolic Pathways ◽  
Coenzyme A ◽  
Protein A ◽  
Biosynthesis Pathway ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Uncharacterized Gene ◽  
Wide Range ◽  
Insight Into

ABSTRACT Dephospho-coenzyme A (dephospho-CoA) kinase (DPCK) catalyzes the ATP-dependent phosphorylation of dephospho-CoA, the final step in coenzyme A (CoA) biosynthesis. DPCK has been identified and characterized in bacteria and eukaryotes but not in archaea. The hyperthermophilic archaeon Thermococcus kodakarensis encodes two homologs of bacterial DPCK and the DPCK domain of eukaryotic CoA synthase, TK1334 and TK2192. We purified the recombinant TK1334 and TK2192 proteins and found that they lacked DPCK activity. Bioinformatic analyses showed that, in several archaea, the uncharacterized gene from arCOG04076 protein is fused with the gene for phosphopantetheine adenylyltransferase (PPAT), which catalyzes the reaction upstream of the DPCK reaction in CoA biosynthesis. This observation suggested that members of arCOG04076, both fused to PPAT and standalone, could be the missing archaeal DPCKs. We purified the recombinant TK1697 protein, a standalone member of arCOG04076 from T. kodakarensis, and demonstrated its GTP-dependent DPCK activity. Disruption of the TK1697 resulted in CoA auxotrophy, indicating that TK1697 encodes a DPCK that contributes to CoA biosynthesis in T. kodakarensis. TK1697 homologs are widely distributed in archaea, suggesting that the arCOG04076 protein represents a novel family of DPCK that is not homologous to bacterial and eukaryotic DPCKs but is distantly related to bacterial and eukaryotic thiamine pyrophosphokinases. We also constructed and characterized gene disruption strains of TK0517 and TK2128, homologs of bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and PPAT, respectively. Both strains displayed CoA auxotrophy, indicating their contribution to CoA biosynthesis. Taken together with previous studies, the results experimentally validate the entire CoA biosynthesis pathway in T. kodakarensis. IMPORTANCE CoA is utilized in a wide range of metabolic pathways, and its biosynthesis is essential for all life. Pathways for CoA biosynthesis in bacteria and eukaryotes have been established. In archaea, however, the enzyme that catalyzes the final step in CoA biosynthesis, dephospho-CoA kinase (DPCK), had not been identified. In the present study, bioinformatic analyses identified a candidate for the DPCK in archaea, which was biochemically and genetically confirmed in the hyperthermophilic archaeon Thermococcus kodakarensis. Genetic analyses on genes presumed to encode bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and phosphopantetheine adenylyltransferase confirmed their involvement in CoA biosynthesis. Taken together with previous studies, the results reveal the entire pathway for CoA biosynthesis in a single archaeon and provide insight into the different mechanisms of CoA biosynthesis and their distribution in nature.

scholarly journals Endophytic Bacterium-Triggered Reactive Oxygen Species Directly Increase Oxygenous Sesquiterpenoid Content and Diversity in Atractylodes lancea

2015 ◽  
Vol 82 (5) ◽  
pp. 1577-1585 ◽  
Author(s):  
Jia-Yu Zhou ◽  
Jie Yuan ◽  
Xia Li ◽  
Yi-Fan Ning ◽  
Chuan-Chao Dai
Keyword(s):  
Reactive Oxygen Species ◽  
Singlet Oxygen ◽  
Oxidative Burst ◽  
Reactive Oxygen ◽  
Endophytic Bacterium ◽  
Enzymatic Oxidation ◽  
Oxygen Species ◽  
Oxidation Reactions ◽  
In Planta ◽  
Content Type

ABSTRACTOxygenous terpenoids are active components of many medicinal plants. However, current studies that have focused on enzymatic oxidation reactions cannot comprehensively clarify the mechanisms of oxygenous terpenoid synthesis and diversity. This study shows that an endophytic bacterium can trigger the generation of reactive oxygen species (ROS) that directly increase oxygenous sesquiterpenoid content and diversity inAtractylodes lancea.A. lanceais a famous but endangered Chinese medicinal plant that contains abundant oxygenous sesquiterpenoids. Geo-authenticA. lanceaproduces a wider range and a greater abundance of oxygenous sesquiterpenoids than the cultivated herb. Our previous studies have shown the mechanisms behind endophytic promotion of the production of sesquiterpenoid hydrocarbon scaffolds; however, how endophytes promote the formation of oxygenous sesquiterpenoids and their diversity is unclear. After colonization byPseudomonas fluorescensALEB7B, oxidative burst and oxygenous sesquiterpenoid accumulation inA. lanceaoccur synchronously. Treatment with exogenous hydrogen peroxide (H2O2) or singlet oxygen induces oxidative burst and promotes oxygenous sesquiterpenoid accumulationin planta. Conversely, pretreatment of plantlets with the ROS scavenger ascorbic acid significantly inhibits the oxidative burst and oxygenous sesquiterpenoid accumulation induced byP. fluorescensALEB7B. Furtherin vitrooxidation experiments show that several oxygenous sesquiterpenoids can be obtained from direct oxidation caused by H2O2or singlet oxygen. In summary, this study demonstrates that endophytic bacterium-triggered ROS can directly oxidize oxygen-free sesquiterpenoids and increase the oxygenous sesquiterpenoid content and diversity inA. lancea, providing a novel explanation of the mechanisms of oxygenous terpenoid synthesisin plantaand an essential complementarity to enzymatic oxidation reactions.

scholarly journals Distinct Modified Nucleosides in tRNATrp from the Hyperthermophilic Archaeon Thermococcus kodakarensis and Requirement of tRNA m2G10/m22G10 Methyltransferase (Archaeal Trm11) for Survival at High Temperatures

2019 ◽  
Vol 201 (21) ◽  
Author(s):  
Akira Hirata ◽  
Takeo Suzuki ◽  
Tomoko Nagano ◽  
Daishiro Fujii ◽  
Mizuki Okamoto ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
High Temperatures ◽  
Modified Nucleosides ◽  
Liquid Chromatography Mass Spectrometry ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Chromatography Mass Spectrometry ◽  
Modified Nucleoside

ABSTRACT tRNA m2G10/m22G10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms N2,N2-dimethylguanosine (m22G10) via N2-methylguanosine (m2G10). We determined the complete sequence of tRNATrp, one of the substrate tRNAs for archaeal Trm11 from Thermococcus kodakarensis, a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNATrp identified 15 types of modified nucleoside at 21 positions. Several modifications were found at novel positions in tRNA, including 2′-O-methylcytidine at position 6, 2-thiocytidine at position 17, 2′-O-methyluridine at position 20, 5,2′-O-dimethylcytidine at position 32, and 2′-O-methylguanosine at position 42. Furthermore, methylwyosine was found at position 37 in this tRNATrp, although 1-methylguanosine is generally found at this location in tRNATrp from other archaea. We constructed trm11 (Δtrm11) and some gene disruptant strains and compared their tRNATrp with that of the wild-type strain, which confirmed the absence of m22G10 and other corresponding modifications, respectively. The lack of 2-methylguanosine (m2G) at position 67 in the trm11 trm14 double disruptant strain suggested that this methylation is mediated by Trm14, which was previously identified as an m2G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures. The m22G10 modification might have effects on stabilization of tRNA and/or correct folding of tRNA at the high temperatures. Collectively, these results provide new clues to the function of modifications and the substrate specificities of modification enzymes in archaeal tRNA, enabling us to propose a strategy for tRNA stabilization of this archaeon at high temperatures. IMPORTANCE Thermococcus kodakarensis is a hyperthermophilic archaeon that can grow at 60 to 100°C. The sequence of tRNATrp from this archaeon was determined by liquid chromatography/mass spectrometry. Fifteen types of modified nucleoside were observed at 21 positions, including 5 modifications at novel positions; in addition, methylwyosine at position 37 was newly observed in an archaeal tRNATrp. The construction of trm11 (Δtrm11) and other gene disruptant strains confirmed the enzymes responsible for modifications in this tRNA. The lack of 2-methylguanosine (m2G) at position 67 in the trm11 trm14 double disruptant strain suggested that this position is methylated by Trm14, which was previously identified as an m2G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures.

scholarly journals Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

2013 ◽  
Vol 79 (7) ◽  
pp. 2209-2217 ◽  
Author(s):  
Xi Wu ◽  
Chong Zhang ◽  
Izumi Orita ◽  
Tadayuki Imanaka ◽  
Toshiaki Fukui ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Enantiomeric Excess ◽  
Chiral Alcohols ◽  
Secondary Alcohols ◽  
Optimal Temperature ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Highly Active ◽  
Thermococcus Kodakarensis Kod1

ABSTRACTA novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeonThermococcus kodakarensisKOD1 (TkADH). The gene,tk0845, which encodes an aldo-keto reductase, was heterologously expressed inEscherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparentKmvalues for the cofactors NAD(P)+and NADPH were similar within a range of 66 to 127 μM.TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at themetaandparapositions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee).TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50%n-hexane orn-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.

scholarly journals The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Yasuyuki Yamamoto ◽  
Tamotsu Kanai ◽  
Tsuyoshi Kaneseki ◽  
Haruyuki Atomi
Keyword(s):  
Amino Acids ◽  
Transcriptional Regulation ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Aromatic Amino Acid Biosynthesis

ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcales aromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.

scholarly journals A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Jian-qiang Jin ◽  
Shin-ichi Hachisuka ◽  
Takaaki Sato ◽  
Tsuyoshi Fujiwara ◽  
Haruyuki Atomi
Keyword(s):  
Lipoic Acid ◽  
Oxidative Decarboxylation ◽  
Biotin Synthase ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Archaeal Species ◽  
Lipoyl Synthase ◽  
H Protein

ABSTRACT Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea. IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea. Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.

scholarly journals Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

2013 ◽  
Vol 80 (3) ◽  
pp. 1108-1115 ◽  
Author(s):  
Nasir Ahmad ◽  
Naeem Rashid ◽  
Muhammad Saleem Haider ◽  
Mehwish Akram ◽  
Muhammad Akhtar
Keyword(s):  
Metal Ions ◽  
Half Life ◽  
Unique Feature ◽  
Acetate Buffer ◽  
Citrate Buffer ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Type Iii ◽  
Ph Range

ABSTRACTA novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeonThermococcus kodakarensis(TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. TheKmvalue toward pullulan was 2 mg ml−1, with aVmaxof 109 U mg−1. Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.

scholarly journals Engineering of the Hyperthermophilic Archaeon Thermococcus kodakarensis for Chitin-Dependent Hydrogen Production

2017 ◽  
Vol 83 (15) ◽  
Author(s):  
Mehwish Aslam ◽  
Ayumi Horiuchi ◽  
Jan-Robert Simons ◽  
Savyasachee Jha ◽  
Masahiro Yamada ◽  
...  
Keyword(s):  
Hydrogen Generation ◽  
Gene Promoter ◽  
Culture Broth ◽  
Surface Glycoprotein ◽  
Glycolytic Flux ◽  
Hydrogen Yield ◽  
Cell Surface Glycoprotein ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis

ABSTRACT Thermococcus kodakarensis is a hyperthermophilic archaeon that harbors a complete set of genes for chitin degradation to fructose 6-phosphate. However, wild-type T. kodakarensis KOD1 does not display growth on chitin. In this study, we developed a T. kodakarensis strain that can grow on chitin via genetic and adaptive engineering. First, a chitinase overproduction strain (KC01) was constructed by replacing the chitinase gene promoter with a strong promoter from the cell surface glycoprotein gene, resulting in increased degradation of swollen chitin and accumulation of N-,N′-diacetylchitobiose in the medium. To enhance N-,N′-diacetylchitobiose assimilation in KC01, genes encoding diacetylchitobiose deacetylase, exo-β-d-glucosaminidase, and glucosamine-6-phosphate deaminase were also overexpressed to obtain strain KC04. To strengthen the glycolytic flux of KC04, the gene encoding Tgr (transcriptional repressor of glycolytic genes) was disrupted to obtain strain KC04Δt. In both KC04 and KC04Δt strains, degradation of swollen chitin was further enhanced. In the culture broth of these strains, the accumulation of glucosamine was observed. KC04Δt was repeatedly inoculated in a swollen-chitin-containing medium for 13 cultures. This adaptive engineering strategy resulted in the isolation of a strain (KC04ΔtM1) that showed almost complete degradation of 0.4% (wt/vol) swollen chitin after 90 h. The strain produced high levels of acetate and ammonium in the culture medium, and, moreover, molecular hydrogen was generated. This strongly suggests that strain KC04ΔtM1 has acquired the ability to convert chitin to fructose 6-phosphate via deacetylation and deamination and further convert fructose 6-phosphate to acetate via glycolysis coupled to hydrogen generation. IMPORTANCE Chitin is a linear homopolymer of β-1,4-linked N-acetylglucosamine and is the second most abundant biomass next to cellulose. Compared to the wealth of research focused on the microbial degradation and conversion of cellulose, studies addressing microbial chitin utilization are still limited. In this study, using the hyperthermophilic archaeon Thermococcus kodakarensis as a host, we have constructed a strain that displays chitin-dependent hydrogen generation. The apparent hydrogen yield per unit of sugar consumed was slightly higher with swollen chitin than with starch. As gene manipulation in T. kodakarensis is relatively simple, the strain constructed in this study can also be used as a parent strain for the development and expansion of chitin-dependent biorefinery, in addition to its capacity to produce hydrogen.

scholarly journals Hyperthermophilic ArchaeonThermococcus kodakarensisUtilizes a Four-Step Pathway for NAD+Salvage through Nicotinamide Deamination

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Shin-ichi Hachisuka ◽  
Takaaki Sato ◽  
Haruyuki Atomi
Keyword(s):  
Nicotinic Acid ◽  
De Novo ◽  
Amide Bond ◽  
Significant Activity ◽  
Genetic Analyses ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Degradation Rates ◽  
Mutant Cells

ABSTRACTMany organisms possess pathways that regenerate NAD+from its degradation products, and two pathways are known to salvage NAD+from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeonThermococcus kodakarensisis supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved inde novobiosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+salvage inT. kodakarensisrequires deamination of Nm and proceeds through the four-step pathway.IMPORTANCEHyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD+from nicotinamide (Nm) in the hyperthermophilic archaeonThermococcus kodakarensis. The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD+itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.

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