Kazunori Ushimaru

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

ABSTRACT In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc.

Characterization of polyhydroxyalkanoate (PHA) synthase derived from Delftia acidovorans DS-17 and the influence of PHA production in Escherichia coli

Heterologous expression of polyhydroxyalkanoate (PHA) synthase from Delftia acidovorans DS-17 (PhaC(Da)) in Escherichia coli JM109 leads to effective production of high-molecular-weight poly[(R)-3-hydroxybutyrate] [P(3HB)]. This study examined the effect of PhaC(Da) expression on P(3HB) production in E. coli JM109 (Da strain) by comparing with the strain expressing PHA synthase (PhaC(Re)) from Ralstonia eutropha (Re strain). First, the kinetic properties of PhaC(Da) were investigated. Among the five detergents examined, Triton X-100 remarkably activated PhaC(Da), as well as PhaC(Re). The affinity of PhaC(Da) for its substrate was lower than that of PhaC(Re), whereas the maximum reaction rate of PhaC(Da) was higher than that of PhaC(Re). However, the kinetic differences were not likely to influence P(3HB) production in the cells. Under conditions of P(3HB) production, the translational levels of monomer-supplying enzymes (PhaA and PhaB) were similar in both the Da and Re strains, whereas PhaC exhibited different expression levels: the abundance of soluble PhaC(Da) was lower than that of soluble PhaC(Re). This observation suggests that the production of high-molecular-weight P(3HB) by the Da strain would be attributed to the low amounts of active PhaC(Da) in the cells.

Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

ABSTRACT The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter−1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter−1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.

Characterization of binding preference of polyhydroxyalkanoate biosynthesis-related multifunctional protein PhaM from Ralstonia eutropha

The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaMRe) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaCRe) was activated by the presence of PhaMRe but PHA synthase from Aeromonas caviae (PhaCAc) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaMRe interacted with PhaCRe directly, but did not interact with PhaCAc. These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaMRe. Further analyses indicated that PhaMRe has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaARe, phaBRe, and phaCRe/phaCAc) and the phaMRe gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaMRe decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaCRe-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaMRe expression was observed in PhaCAc-expressing E. coli. These observations suggest that PhaMRe affects cellular physiology through the PhaM-PhaC interaction.

Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

Recombinant Ralstonia eutropha strain PHB(-)4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB(-)4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB(-)4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.

Real-Time Observation of Enzymatic Polyhydroxyalkanoate Polymerization Using High-Speed Scanning Atomic Force Microscopy

The initial stage of in vitro polyhydroxyalkanoate (PHA) polymerization by PHA synthase from Ralstonia eutropha (PhaCRe) on a mica substrate in water was observed using high-speed scanning atomic force microscopy (HS-AFM). Before PHA polymerization, the adsorption–desorption cycle of the PhaCRe molecule on mica was observed in real time. For approximately 30 s after the addition of the PHA monomer, no significant change was observed on the mica substrate, but PhaCRe could be transformed into an active enzyme in water upon contact with the monomer during this period. Subsequently, linearly elongating rod-shaped objects were observed on the mica substrate, plausibly as a result of the polymerization reaction. The height of these elongating objects was considerably larger than the expected height for a single PHA chain. This observation suggests that PHA chains generated during the reported experiments might form some kind of a semiregular structure.

Biodegradable and magnetic core–shell composite particle prepared by emulsion solvent diffusion method

The present paper describes optimization of preparation conditions of a core–shell composite particle, and its heat generation by alternating magnetic fields. The composite particles are prepared with a modified emulsion solvent diffusion method, which is combined with Pickering emulsion stabilized by magnetic nanoparticles. In this method, the magnetic nanoparticles act as an emulsifier, and its amount and size are crucial to morphology of the composite particles. The magnetic nanoparticles of 8–9 nm would be strongly adsorbed at a liquid–liquid interface rather than the larger nanoparticles. At the optimized concentration of the magnetic nanoparticles suspension for the preparation, small and uniform composite particles are obtained since the amount of the nanoparticles is enough to prevent coalescence of droplets during the formation of the composites. The heat generation by alternating magnetic fields emerged certainly. This result suggests the composite particles have a property as a heat-generating carrier for hyperthermia treatment.