Izumi Orita

The Ribulose Monophosphate Pathway Substitutes for the Missing Pentose Phosphate Pathway in the Archaeon Thermococcus kodakaraensis

The ribulose monophosphate (RuMP) pathway, involving 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is now recognized as a widespread prokaryotic pathway for formaldehyde fixation and detoxification. Interestingly, HPS and PHI homologs are also found in a variety of archaeal strains, and recent biochemical and genome analyses have raised the possibility that the reverse reaction of formaldehyde fixation, i.e., ribulose 5-phosphate (Ru5P) synthesis from fructose 6-phosphate, may function in the biosynthesis of Ru5P in some archaeal strains whose pentose phosphate pathways are imperfect. In this study, we have taken a genetic approach to address this possibility by using the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. This strain possesses a single open reading frame (TK0475) encoding an HPS- and PHI-fused protein. The recombinant HPS-PHI-fused enzyme exhibited the expected HPS and PHI activities in both directions (formaldehyde fixing and Ru5P synthesizing). The TK0475 deletion mutant Delta hps-phi-7A did not exhibit any growth in minimal medium, while growth of the mutant strain could be recovered by the addition of nucleosides to the medium. This auxotrophic phenotype together with the catalytic properties of the HPS-PHI-fused enzyme reveal that HPS and PHI are essential for the biosynthesis of Ru5P, the precursor of nucleotides, showing that the RuMP pathway is the only relevant pathway for Ru5P biosynthesis substituting for the classical pentose phosphate pathway missing in this archaeon.

Bifunctional enzyme fusion of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase

The formaldehyde-fixing enzymes, 3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), are the key enzymes catalyzing sequential reactions in the ribulose monophosphate (RuMP) pathway. In this study, we generated two fused gene constructs of the hps and phi genes (i.e., hps–phi and phi–hps) from a methylotrophic bacterium Mycobacterium gastri MB19. The gene product of hps–phi exhibited both HPS and PHI activities at room temperature and catalyzed the sequential reactions more efficiently than a simple mixture of the individual enzymes. The gene product of phi–hps failed to display any enzyme activity. Escherichia coli strains harboring the hps–phi gene consumed formaldehyde more efficiently and exhibited better growth in a formaldehyde-containing medium than the host strain. Our results demonstrate that the engineered fusion gene has the possibility to be used to establish a formaldehyde-resistance detoxification system in various organisms.

Assimilation of Formaldehyde in Transgenic Plants Due to the Introduction of the Bacterial Ribulose Monophosphate Pathway Genes

Formaldehyde (HCHO) is an air pollutant suspected of being carcinogenic and a cause of sick-house syndrome. Microorganisms called methylotrophs, which can utilize reduced C1 compounds such as methane and methanol, fix and assimilate HCHO, whereas most plants are unable to assimilate HCHO directly. We found that a bacterial formaldehyde-fixing pathway (ribulose monophosphate pathway) can be integrated as a bypass to the Calvin-Benson cycle in transgenic Arabidopsis thaliana and tobacco by genetic engineering. These plants showed enhanced tolerance to HCHO and enhanced capacity to eliminate gaseous HCHO by fixing it as a sugar phosphate. Our results provide a novel strategy for phytoremediation of HCHO pollution, and also represent the first step toward the production of plants that can assimilate natural gas-derived C1 compounds.

Characterization and Functional Analyses of R-Specific Enoyl Coenzyme A Hydratases in Polyhydroxyalkanoate-Producing Ralstonia eutropha

ABSTRACT A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R-specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded by phaJ4 from Pseudomonas aeruginosa (phaJ4Pa ). The recombinant forms of the three proteins, termed PhaJ4a Re to PhaJ4c Re , actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C4 to C8. PhaJ4a Re and PhaJ4b Re showed >10-fold-higher catalytic efficiency than PhaJ4c Re . The functions of the new PhaJ4 proteins were investigated using previously engineered R. eutropha strains as host strains; these strains are capable of synthesizing poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Deletion of phaJ4aRe from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4bRe or phaJ4cRe , indicating that only PhaJ4a Re was one of the major enzymes supplying the (R)-3HHx-CoA monomer through β-oxidation. Introduction of phaJ4aRe or phaJ4bRe into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4cRe did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co-3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially when phaJ4aRe or phaJ4bRe was tandemly introduced with phaJAc from Aeromonas caviae.

The Archaeon Pyrococcus horikoshii Possesses a Bifunctional Enzyme for Formaldehyde Fixation via the Ribulose Monophosphate Pathway

Pyrococcus horikoshii OT3, a hyperthermophilic and anaerobic archaeon, was found to have an open reading frame (PH1938) whose deduced amino acid sequence of the N-terminal and C-terminal halves showed significant similarity to two key enzymes of the ribulose monophosphate pathway for formaldehyde fixation in methylotrophic bacteria, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), respectively. The organism constitutively produced the encoded protein and exhibited activity of the sequential HPS- and PHI-mediated reactions in a particulate fraction. The full-length gene encoding the hybrid enzyme, the sequence corresponding to the HPS region, and the sequence corresponding to the PHI region were expressed in Escherichia coli and were found to produce active enzymes, rHps-Phi, rHps, or rPhi, respectively. Purified rHps-Phi and rHps were found to be active at the growth temperatures of the parent strain, but purified rPhi exhibited significant susceptibility to heat, suggesting that thermostability of the PHI moiety of the bifunctional enzyme (rHps-Phi) resulted from fusion with HPS. The bifunctional enzyme catalyzed the sequential reaction much more efficiently than a mixture of rHps and rPhi. These and other biochemical characterizations of the PH1938 gene product suggest that the ribulose monophosphate pathway plays a significant role in the archaeon under extreme environmental conditions.

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by Plac, Ptac, or PBAD derived from Escherichia coli, or promoter regions of phaC1 (PphaC) or phaP1 (PphaP) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. Plac, Ptac, PphaC, and PphaP mediated constitutive gene expression, among which Ptac was the strongest promoter. lacI-Ptac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-PBAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-PphaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-PphaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.

Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions

Methylobacterium extorquens AM1 has been shown to accumulate polyhydroxyalkanoate (PHA) composed solely of (R)-3-hydroxybutyrate (3HB) during methylotrophic growth. The present study demonstrated that the wild-type strain AM1 grown under Co2+-deficient conditions accumulated copolyesters of 3HB and a C5-monomer, (R)-3-hydroxyvalerate (3HV), using methanol as the sole carbon source. The 3HV unit was supposed to be derived from propionyl-CoA, synthesized via the ethylmalonyl-CoA pathway impaired by Co2+ limitation. This assumption was strongly supported by the dominant incorporation of the 3HV unit into PHA when a strain lacking propionyl-CoA carboxylase was incubated with methanol. Further genetic engineering of M. extorquens AM1 was employed for the methylotrophic synthesis of PHA copolymers. A recombinant strain of M. extorquens AM1CAc in which the original PHA synthase gene phaCMe had been replaced by phaCAc, encoding an enzyme with broad substrate specificity from Aeromonas caviae, produced a PHA terpolymer composed of 3HB, 3HV, and a C6-monomer, (R)-3-hydroxyhexanoate, from methanol. The cellular content and molecular weight of the PHA accumulated in the strain AM1CAc were higher than those of PHA in the wild-type strain. The triple deletion of three PHA depolymerase genes in M. extorquens AM1CAc showed no significant effects on growth and PHA biosynthesis properties. Overexpression of the genes encoding β-ketothiolase and NADPH-acetoacetyl-CoA reductase increased the cellular PHA content and 3HV composition in PHA, although the cell growth on methanol was decreased. This study opens up the possibility of producing practical PHA copolymers with methylotrophic bacteria using methanol as a feedstock.

Overexpression of an HPS/PHI fusion enzyme from Mycobacterium gastri in chloroplasts of geranium enhances its ability to assimilate and phytoremediate formaldehyde.

Abstract3-Hexulose-6-phosphate synthase (HPS) and 6-phosphate-3-hexuloisomerase (PHI) are two key enzymes in the formaldehyde (HCHO) assimilation pathway in methylotrophs. The HPS/PHI fusion protein, encoded by the chimeric gene of hps and phi from Mycobacterium gastri MB19, possesses both HPS and PHI activities in an Escherichia coli transformant. Overexpression of the fusion protein in chloroplasts of geranium (Pelargonium sp. Frensham) created a photosynthetic HCHO assimilation pathway according to 13C-NMR analysis. The transgenic plants exhibited an enhanced ability in HCHO-uptake and [14C]HCHO-assimilation. Moreover, the transgenic plants showed greater HCHO-resistance and stronger capacity in purification of the HCHO-polluted air. Therefore, the use of the single chimeric gene may not only greatly simplify the transformation procedure but also improve the efficiency of phytoremediating HCHO in ornamental plants.

Application of a novel thermostable NAD(P)H oxidase from hyperthermophilic archaeon for the regeneration of both NAD+ and NADP+

A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkNOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of TkNOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP+ and NAD+ in alcohol dehydrogenase (ADH)‐catalyzed enantioselective oxidation of racemic 1‐phenylethanol. NADP+ regeneration at 30°C was performed by TkNOX coupled with (R)‐specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)‐1‐phenylethanol. The use of TkNOX with moderately thermostable (S)‐specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD+ regeneration at high temperatures. Optically pure (R)‐1‐phenylethanol was successfully obtained by this system after a shorter reaction time at 45–60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of TkNOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air. Biotechnol. Bioeng. 2012;109: 53–62.

Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16

This study describes metabolite profiles of Ralstonia eutropha H16 focusing on biosynthesis of polyhydroxyalkanoates (PHAs), bacterial polyesters attracted as biodegradable bio-based plastics. As CoA-thioesters are important intermediates in PHA biosynthesis, four kinds of acyl-CoAs with medium chain length were prepared and used to establish analytical conditions for capillary electrophoresis-electron spray ionization-tandem mass spectrometry (CE–ESI-MS/MS). Metabolites were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose and PHA production phase on octanoate, and subjected to stable isotope dilution-based comparative quantification by multiple reaction monitoring using CE–ESI-MS/MS and 13C-labeled metabolites prepared by extraction from R. eutropha mutant grown on U-13C6-glucose. This procedure allowed to quantify relative changes of 94 ionic metabolites including CoA-thioesters. Hexose-phosphates except for glucose 1-phosphate were decreased in the PHA production phase than in the growth phase, suggesting reduced flux of sugar degradation after the cell growth. Several intermediates in TCA cycle and gluconeogenesis were increased in the PHA production phase on octanoate. Interestingly, ribulose 1,5-bisphosphate were detected in all the samples examined, raising possibilities of CO2 fixation by Calvin–Benson–Bassham cycle in this bacterium even under heterotrophic growth conditions. Turnover of acyl moieties through β-oxidation was suggested to be active on fructose, as CoA-thioesters of C6 and C8 were detected in the fructose-grown cells. In addition, major metabolic pools in R. eutropha cells were estimated from the signal intensities. The results of the present study provided new insights into global metabolisms in PHA-producing R. eutropha.