Kei Motomura

Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate

ABSTRACT Heat-treated Escherichia coli producing Thermus polyphosphate kinase regenerated ATP by using exogenous polyphosphate. This recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. In this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and polyphosphate.

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

ABSTRACT Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by “high-energy” phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.

The silica-binding Si-tag functions as an affinity tag even under denaturing conditions

We recently reported a one-step affinity purification method using a silica-binding protein, designated Si-tag, as a fusion partner and silica particles as the specific adsorbents (Ikeda et al., Protein Expr. Purif. 71 [2010] 91-95) [13]. In this study, we demonstrate that the Si-tag also binds to the silica surface even under denaturing conditions, thereby facilitating affinity purification of recombinant proteins from inclusion bodies. A fusion protein of the Si-tag and a biotin acceptor peptide (AviTag), which was expressed as inclusion bodies in Escherichia coli, was used as a model protein. To simplify our purification method, we disrupted recombinant E. coli cells by sonication in the presence of 8M urea with concomitant solubilization of the inclusion bodies. The fusion protein was recovered with a purity of 90 ± 3% and yield of 92 ± 6% from the cleared cell lysate. We also discuss the binding mechanism of the Si-tag to a silica surface in the presence of high concentrations of denaturant. We propose that the intrinsic disorder of the polycationic Si-tag polypeptide plays an important role in its binding to the silica surface under denaturing conditions.

Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506

Phosphite dehydrogenase (PtxD), which catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH, has great potential for NADH regeneration in industrial biocatalysts. Here, we isolated a soil bacterium, Ralstonia sp. strain 4506, that grew at 45°C on a minimal medium containing phosphite as the sole source of phosphorus. A recombinant PtxD of Ralstonia sp. (PtxD(R4506)) appeared in the soluble fraction in Escherichia coli. The purified PtxD(R4506) showed 6.7-fold greater catalytic efficiency (V(max)/K(m)) than the first characterized PtxD of Pseudomonas stutzeri (PtxD(PS)). Moreover, the purified PtxD(R4506) showed maximum activity at 50°C, and its half-life of thermal inactivation at 45°C was 80.5h, which is approximately 3,450-fold greater than that of PtxD(PS). Therefore, we concluded that PtxD(R4506), which shows high catalytic efficiency, solubility, and thermostability, would be useful for NADH regeneration applications.

Application of a phosphite dehydrogenase gene as a novel dominant selection marker for yeasts

The use of antibiotic resistance markers in the commercial application of genetically modified microorganisms is limited due to restrictions on the release of antibiotics and their resistance genes to the environment. To avoid contamination by other microorganisms, the development of a dominant selection marker with low environmental risks is still needed. Here we demonstrated a new selection system for Schizosaccharomyces pombe and Saccharomyces cerevisiae using a bacterial phosphite dehydrogenase gene (ptxD). A Sz. pombe transformant carrying ptxD under a strong promoter or on a multicopy plasmid grew on a minimal medium containing phosphite (Pt) as a sole source of phosphorus. To adapt this system to S. cerevisiae strains, codon optimization of ptxD was necessary. The codon-optimized ptxD system appeared effective in not only laboratorial but also industrial S. cerevisiae strains that are diploid or polyploid. Since Pt is a safe and inexpensive chemical, ptxD could be used as a novel dominant selection marker applicable on an industrial scale.

Stable polyphosphate accumulation by a pseudo-revertant of an Escherichia coli phoU mutant

AbstractphoU mutants of bacteria are potentially useful for the removal of inorganic phosphate (Pi) from sewage because they can accumulate a large amounts of polyphosphate (polyP). However, the growth of phoU mutants is severely defective and is easily outgrown by revertant(s) that have lost the ability to accumulate polyP during growth in a nutrient-rich medium. We found that a pseudo-revertant, designated LAP[+], that appeared in a culture of an Escherichia coli phoU mutant that could accumulate polyP even after ten serial passages. Reduction in the expression of the Pi-specific transporter Pst in LAP[+] may contribute to relieving stresses such as excess Pi incorporation that could stimulate reversions. The discovery of a LAP[+] provides a clue to generate phoU mutants that accumulate polyP in a stable manner.

A Novel Biocontainment Strategy Makes Bacterial Growth and Survival Dependent on Phosphite

There is a growing demand to develop biocontainment strategies that prevent unintended proliferation of genetically modified organisms in the open environment. We found that the hypophosphite (H3PO2, HPt) transporter HtxBCDE from Pseudomonas stutzeri WM88 was also capable of transporting phosphite (H3PO3, Pt) but not phosphate (H3PO4, Pi), suggesting the potential for engineering a Pt/HPt-dependent bacterial strain as a biocontainment strategy. We disrupted all Pi and organic Pi transporters in an Escherichia coli strain expressing HtxABCDE and a Pt dehydrogenase, leaving Pt/HPt uptake and oxidation as the only means to obtain Pi. Challenge on non-permissive growth medium revealed that no escape mutants appeared for at least 21 days with a detection limit of 1.94 × 10−13 per colony forming unit. This represents, to the best of our knowledge, the lowest escape frequency among reported strategies. Since Pt/HPt are ecologically rare and not available in amounts sufficient for the growth of the Pt/HPt-dependent bacteria, this strategy offers a reliable and practical method for biocontainment.

The C-Terminal Zwitterionic Sequence of CotB1 Is Essential for Biosilicification of the Bacillus cereus Spore Coat

UNLABELLED Silica is deposited in and around the spore coat layer of Bacillus cereus, and enhances the spores acid resistance. Several peptides and proteins, including diatom silaffin and silacidin peptides, are involved in eukaryotic silica biomineralization (biosilicification). Homologous sequence search revealed a silacidin-like sequence in the C-terminal region of CotB1, a spore coat protein of B. cereus. The negatively charged silacidin-like sequence is followed by a positively charged arginine-rich sequence of 14 amino acids, which is remarkably similar to the silaffins. These sequences impart a zwitterionic character to the C terminus of CotB1. Interestingly, the cotB1 gene appears to form a bicistronic operon with its paralog, cotB2, the product of which, however, lacks the C-terminal zwitterionic sequence. A ΔcotB1B2 mutant strain grew as fast and formed spores at the same rate as wild-type bacteria but did not show biosilicification. Complementation analysis showed that CotB1, but neither CotB2 nor C-terminally truncated mutants of CotB1, could restore the biosilicification activity in the ΔcotB1B2 mutant, suggesting that the C-terminal zwitterionic sequence of CotB1 is essential for the process. We found that the kinetics of CotB1 expression, as well as its localization, correlated well with the time course of biosilicification and the location of the deposited silica. To our knowledge, this is the first report of a protein directly involved in prokaryotic biosilicification. IMPORTANCE Biosilicification is the process by which organisms incorporate soluble silicate in the form of insoluble silica. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification was not studied until recently. We previously demonstrated that biosilicification occurs in Bacillus cereus and its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acid. The present study reveals that a B. cereus spore coat protein, CotB1, which carried a C-terminal zwitterionic sequence, is essential for biosilicification. Our results provide the first insight into mechanisms required for biosilicification in prokaryotes.

Biological Phosphite Oxidation and Its Application to Phosphorus Recycling

Several chemical industrial processes produce phosphite (Pt) as a by-product. This Pt waste should be recycled and reused as an alternative phosphorus (P) source to reduce the demand for nonrenewable phosphate rock reserves. Nearly all organisms require inorganic phosphate (Pi) or its esters as their P source. Therefore, Pt has been considered a biologically inert P compound, hampering the development of biotechnologies that utilize the Pt waste. During the last decade, the molecular mechanisms involved in the metabolism of inorganic reduced P compounds, including Pt, have been elucidated. Pt dehydrogenase (PtxD) catalyzes the oxidation of Pt to Pi, with a concomitant reduction of NAD+ to NADH, and thus is a promising biocatalyst for developing Pt-based applications. The initial discovery of PtxD, followed by the finding and development of PtxD enzymes with high catalytic activity and thermostability, facilitated the development of several unique biotechnological applications. These applications include (i) a dominant selective cultivation system for microorganisms and plants, (ii) a biological containment strategy for the safe use of genetically modified organisms, and (iii) a cofactor regeneration system for efficient production of chiral compounds by dehydrogenases. This section describes the emerging biotechnology applications that should contribute to the utilization of Pt as a valuable chemical.

Degradation of phosphate polymer polyP enhances lactic fermentation in mice

In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a “molecular fossil” due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.