Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaC for the Biosynthesis of Poly[()-3-hydroxybutyrate--6-hydroxyhexanoate].

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaC was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaC were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx).

Isolation and characterization of polyhydroxyalkanoate-degrading bacteria in seawater at two different depths from Suruga Bay.

Polyhydroxyalkanoate (PHA) is a highly biodegradable microbial polyester, even in marine environments. In this study, we incorporated an enrichment culture-like approach in the process of isolating marine PHA-degrading bacteria. The resulting 91 isolates were suggested to fall into five genera (, , , , and ) based on 16S rRNA analysis, including two novel genera ( and ) as marine PHA-degrading bacteria.

Ethylene glycol metabolism in the poly(ethylene terephthalate)-degrading bacterium Ideonella sakaiensis.

Poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis produces hydrolytic enzymes that convert PET, via mono(2-hydroxyethyl) terephthalate (MHET), into the monomeric compounds, terephthalic acid (TPA), and ethylene glycol (EG). Understanding PET metabolism is critical if this bacterium is to be engineered for bioremediation and biorecycling. TPA uptake and catabolism in I.

A Lipoate-Protein Ligase Is Required for Lipoyl-Protein Biosynthesis in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Lipoic acid is an organosulfur cofactor essential for several key enzyme complexes in oxidative and one-carbon metabolism. It is covalently bound to the lipoyl domain of the E2 subunit in some 2-oxoacid dehydrogenase complexes and the H-protein in the glycine cleavage system. Lipoate-protein ligase (Lpl) is involved in the salvage of exogenous lipoate and attaches free lipoate to the E2 subunit or the H-protein in an ATP-dependent manner.

Development of a Targeted Gene Disruption System in the Poly(Ethylene Terephthalate)-Degrading Bacterium Ideonella sakaiensis and Its Applications to PETase and MHETase Genes.

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of in bioremediation and biorecycling, approaches to studying this bacterium remain limited.

TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Members of harbor a number of genes encoding putative aminotransferase class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon The TK1211 gene was expressed in under the control of the strong, constitutive promoter of the cell surface glycoprotein gene TK0895 (P ). The purified protein did not display aminotransferase activity but exhibited racemase activity.

A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C 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.

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD Salvage through Nicotinamide Deamination.

Many 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.

An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon .

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon We overexpressed the TK2101 gene in and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity.

Metabolism Dealing with Thermal Degradation of NAD in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

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 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.

An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism.

Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB.

Structure and function of an ancestral-type β-decarboxylating dehydrogenase from Thermococcus kodakarensis.

β-Decarboxylating dehydrogenases, which are involved in central metabolism, are considered to have diverged from a common ancestor with broad substrate specificity. In a molecular phylogenetic analysis of 183 β-decarboxylating dehydrogenase homologs from 84 species, TK0280 from Thermococcus kodakarensis was selected as a candidate for an ancestral-type β-decarboxylating dehydrogenase. The biochemical characterization of recombinant TK0280 revealed that the enzyme exhibited dehydrogenase activities toward homoisocitrate, isocitrate, and 3-isopropylmalate, which correspond to key reactions involved in the lysine biosynthetic pathway, tricarboxylic acid cycle, and leucine biosynthetic pathway, respectively.