Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaC_AR, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaC_AR was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent l-valine depletion caused by dominant l-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent d-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent l-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaC_AR⁺, ldhA_Cd-hadA_Cd⁺ encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaC_AR. The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaC_AR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell.

Key points

(R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha(R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identifiedThe engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB Graphical Abstract

DOI： 10.1007/s00253-023-12797-6

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Other Link： https://link.springer.com/article/10.1007/s00253-023-12797-6/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is a biodegradable plastic characterized by high flexibility, softness, a wide process window, and marine biodegradability. PHBHHx is usually produced from structurally related carbon sources, such as vegetable oils or fatty acids, but not from inexpensive carbon sources such as sugars. In previous studies, we demonstrated that engineered strains of a hydrogen-oxidizing bacterium, Cupriavidus necator, synthesized PHBHHx with a high cellular content not only from sugars but also from CO2 as the sole carbon source in the flask culture. In this study, the highly efficient production of PHBHHx from CO2 was investigated via pH-stat jar cultivation of recombinant C. necator strains while feeding the substrate gas mixture (H2/O2/CO2 = 80:10:10 v/v%) to a complete mineral medium in a recycled-gas, closed-circuit culture system. As a result, the dry cell mass and PHBHHx concentration with the strain MF01/pBPP-ccrMeJAc-emd reached up to 59.62 ± 3.18 g·L−1 and 49.31 ± 3.14 g·L−1, respectively, after 216 h of jar cultivation with limited addition of ammonia and phosphate solutions. The 3HHx composition was close to 10 mol%, which is suitable for practical applications. It is expected that the autotrophic cultivation of the recombinant C. necator can be feasible for the mass production of PHBHHx from CO2.

DOI： 10.3390/bioengineering10111304

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BACKGROUND: This study aimed to isolate a novel thermotolerant bacterium that is capable of synthesizing polyhydroxyalkanoate from glycerol under high temperature conditions. RESULTS: A newly thermotolerant polyhydroxyalkanoate (PHA) producing bacterium, Cupriavidus sp. strain CB15, was isolated from corncob compost. The potential ability to synthesize PHA was confirmed by detection of PHA synthase (phaC) gene in the genome. This strain could produce poly(3-hydroxybutyrate) [P(3HB)] with 0.95 g/L (PHA content 75.3 wt% of dry cell weight 1.24 g/L) using glycerol as a carbon source. The concentration of PHA was enhanced and optimized based on one-factor-at-a-time (OFAT) experiments and response surface methodology (RSM). The optimum conditions for growth and PHA biosynthesis were 10 g/L glycerol, 0.78 g/L NH4Cl, shaking speed at 175 rpm, temperature at 45 °C, and cultivation time at 72 h. Under the optimized conditions, PHA production was enhanced to 2.09 g/L (PHA content of 74.4 wt% and dry cell weight of 2.81 g/L), which is 2.12-fold compared with non-optimized conditions. Nuclear magnetic resonance (NMR) analysis confirmed that the extracted PHA was a homopolyester of 3-hydyoxybutyrate. CONCLUSION: Cupriavidus sp. strain CB15 exhibited potential for cost-effective production of PHA from glycerol.

DOI： 10.1186/s12934-023-02059-5

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Publishing type：Research paper (scientific journal)   Publisher：Japan Petroleum Institute  

DOI： 10.1627/jpi.65.213

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Post-transcriptional modifications have critical roles in tRNA stability and function1-4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2'-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2'-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.

DOI： 10.1038/s41586-022-04677-2

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Language：English  

The authors wish to make the following corrections to this paper [...].

DOI： 10.3390/microorganisms10030529

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse β-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

DOI： 10.3390/microorganisms10010184

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Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is a practical kind of bacterial polyhydroxyalkanoates (PHAs). A previous study has established an artificial pathway for the biosynthesis of P(3HB-co-3HHx) from structurally unrelated sugars in Ralstonia eutropha, in which crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) are a key combination for generation of butyryl-CoA and the following chain elongation. This study focused on the installation of the artificial pathway into Escherichia coli. The recombinant strain of E. coli JM109 harboring 11 heterologous genes including Ccr and Emd produced P(3HB-co-3HHx) composed of 14 mol% 3HHx with 41 wt% of dry cellular weight from glucose. Further investigations revealed that the C6 monomer (R)-3HHx-CoA was not supplied by (R)-specific reduction of 3-oxohexanoyl-CoA but by (R)-specific hydration of 2-hexenoyl-CoA formed through reverse β-oxidation after the elongation from C4 to C6. While contribution of the reverse β-oxidation to the conversion of the C4 intermediates was very limited, crotonyl-CoA, a precursor of butyryl-CoA, was generated by dehydration of (R)-3HB-CoA. Several modifications previously reported for enhancement of bioproduction in E. coli were examined for the copolyester synthesis. Elimination of the global regulator Cra or PdhR as well as the block of acetate formation resulted in poor PHA synthesis. The strain lacking RNase G accumulated more PHA but with almost no 3HHx unit. Introduction of the phosphite oxidation system for regeneration of NADPH led to copolyester synthesis with the higher cellular content and higher 3HHx composition by two-stage cultivation with phosphite than those in the absence of phosphite.

DOI： 10.3389/fbioe.2022.888973

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The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through β-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogen-oxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO2 as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H2/O2/CO2 = 8:1:1). The results of GC and 1H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO2 with high cellular content. When 1.0 g/L (NH4)2SO4 was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01∆B1/pBBP-ccrMeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPP-ccrMeJAc-emd harboring short-chain-length-specific PhaJ.

DOI： 10.3390/bioengineering8110179

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Chemolithoautotrophic bacterium Ralstonia eutropha is a versatile host for production of various useful compounds including polyhydroxyalkanoates (PHAs) under both heterotrophic and autotrophic conditions. In this bacterium, Calvin-Benson-Bassham (CBB) cycle is functional even under heterotrophic conditions on sugars and reutilizes CO2 emitted through sugar metabolisms into PHA, leading to increase in yield of the storage polyester. This study focused on isopropanol production from glucose by engineered strains of R. eutropha. The isopropanol-producing strains were constructed by introduction of codon-optimized genes of acetoacetate decarboxylase (adc) and primary-secondary alcohol dehydrogenase (adh) from clostridia into glucose-utilizing and PHA-negative (ΔphaC1) strain of R. eutropha. Several genetic modifications showed that high expression of the isopropanol synthesis genes by using a strong synthetic promoter and deletion of NAD+-dependent (S)-3-hydroxybutyryl-CoA dehydrogenase genes (paaH1 and had) in addition to NADPH-dependent acetoacetyl-CoA reductase genes (phaB1 and phaB3) were effective for improving isopropanol production with low by-production of acetone. Isopropanol titer of 4.13 g/L was achieved by two-stage cultivation of the strain IP-007/pBj5c2-adh-adc, corresponding to overall yield of 0.6 mol mol-glucose-1. The fixation of sugar-derived CO2 during isopropanol synthesis was evaluated by 13C-labelling of the isopropanol produced from [1-13C]-glucose. The 13C-abundance in isopropanol synthesized by the engineered strain was significantly increased up to 4.8%, demonstrating actual reassimilation of CO2 emitted from glucose moiety by decarboxylation and potential contribution towards increase in the carbon yield of isopropanol on glucose.

DOI： 10.1016/j.jbiosc.2021.08.004

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Poly(ethylene terephthalate) (PET) is a widely used plastic in bottles and fibers; its waste products pollute the environment owing to its remarkable durability. Recently, Ideonella sakaiensis 201-F6 was isolated as a unique bacterium that can degrade and assimilate PET, thus paving the way for the bioremediation and bioconversion of PET waste. We found that this strain harbors a poly(hydroxyalkanoate) (PHA) synthesis gene cluster, which is highly homologous with that of Cupriavidus necator, an efficient PHA producer. Cells grown on PET accumulated intracellular PHA at high levels. Collectively, our findings in this study demonstrate that I. sakaiensis can mediate the direct conversion of non-biodegradable PET into environment-friendly plastic, providing a new approach for PET recycling.

DOI： 10.1038/s41598-021-99528-x

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Methylotrophic bacterium Methylorubrum extorquens is a promising microorganism for the production of value-added compounds from methanol. This study focused on the development of a single-cell level biosensor system that detects methanol by using the intrinsic regulatory machinery which responds to the presence of methanol in this bacterium. A green fluorescent protein (GFP) gene located downstream of the promoter region of the serine glyoxylate aminotransferase gene (Psga) or the methanol dehydrogenase subunit 1 precursor gene (PmxaF) was inserted into the chromosome of M. extorquens wild-type strain AM1. The expression of GFP upon methanol exposure was measured by spectrofluorometer and fluorescence-activated cell sorting (FACS). The strain harboring Psga-gfp emitted fluorescence only when methanol was supplied to the culture medium, while the other strain harboring PmxaF-gfp showed high basal fluorescence even in the absence of methanol. The fluorescence intensity of the Psga-gfp strain depended on a methanol concentration higher than 25 μM, and the sensitivity and dose-dependency of this strain were much higher than previous systems using Escherichia coli. The methanol-sensing properties of the engineered M. extorquens strain were comparable to those of a methylotrophic yeast-based biosensor, suggesting the usefulness of methylotrophic microorganisms as platforms for single-cell sensing of C1 compounds. The constructed methanol sensor strain, coupled with flow cytometry techniques, provides a high-throughput and highly sensitive screening method for the selection of functional methanol-producing enzymes.

DOI： 10.1016/j.jbiosc.2021.05.002

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Glycogen synthesis in bacteria is mainly organized by the products of glgB, glgC, and glgA genes comprising the widely known glg operon. On the genome of extremely halophilic archaeon Haloarcula japonica, there was a gene cluster analogous to the bacterial glg operon. In this study, we focused on a GlgC homolog of Ha. japonica, and its recombinant enzyme was prepared and characterized. The enzyme showed highest activity toward GTP and glucose-1-phosphate as substrates in the presence of 2.6 m KCl and predicted to be work as "GDP-glucose pyrophosphorylase" in Ha. japonica.

DOI： 10.1093/bbb/zbab050

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This study investigates the effect of strategies on poly(3-hydroxybutyrate) [P(3HB)] production in bioreactor. In the production of P(3HB), urea and glucose feeding streams were developed to characterize the fed-batch culture conditions for new Cupriavidus necator NSDG-GG mutant. Feeding urea in repeated fed-batch stage (RFB-I) at 6, and 12 h in cultivation led to insignificant kinetic effect on the cell dry mass (CDM) and P(3HB) accumulation. Feeding glucose in repeated fed-batch stage (RFB-II) demonstrated that the incremental feeding approach of glucose after urea in fill-and-draw (F/D) mode at 24, 30, 36, 42, and 48 h in fermentation increased CDM and P(3HB) concentration. In the 1st cycle in RFB-II, the cumulative CDM reached the value of 26.22 g/L and then it increased with the successive repeated fed-batches to attain biomass of 145 g/L at the end of 5th cycle of RFB-II. The final cumulative P(3HB) concentration at the end of 5th cycle of RFB-II reached 111 g/L with the overall yield of 0.50 g P(3HB) g gluc- 1; the CDM productivity from the RFB-II cycles was in the range of 0.84-1.3 g/(L·h). The RFB-II of glucose in an increment mode produced nearly 2.2 times more increase in CDM and P(3HB) productivities compared to the decrement RFB-II mode. Repeated cultivation had also the advantage of avoiding extra time required for innoculum preparation, and sterilization of bioreactor during batch, thereby it increased the overall industrial importance of the process.

DOI： 10.1016/j.jbiotec.2019.10.013

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is a bacterial polyester with high biodegradability, even in marine environments. Ralstonia eutropha has been engineered for the biosynthesis of P(3HB-co-3HHx) from vegetable oils, but its production from structurally unrelated carbon sources remains unsatisfactory. RESULTS: Ralstonia eutropha strains capable of synthesizing P(3HB-co-3HHx) from not only fructose but also glucose and glycerol were constructed by integrating previously established engineering strategies. Further modifications were made at the acetoacetyl-CoA reduction step determining flux distribution responsible for the copolymer composition. When the major acetoacetyl-CoA reductase (PhaB1) was replaced by a low-activity paralog (PhaB2) or enzymes for reverse β-oxidation, copolyesters with high 3HHx composition were efficiently synthesized from glucose, possibly due to enhanced formation of butyryl-CoA from acetoacetyl-CoA via (S)-3HB-CoA. P(3HB-co-3HHx) composed of 7.0 mol% and 12.1 mol% 3HHx fractions, adequate for practical applications, were produced at cellular contents of 71.4 wt% and 75.3 wt%, respectively. The replacement by low-affinity mutants of PhaB1 had little impact on the PHA biosynthesis on glucose, but slightly affected those on fructose, suggesting altered metabolic regulation depending on the sugar-transport machinery. PhaB1 mostly acted in the conversion of acetoacetyl-CoA when the cells were grown on glycerol, as copolyester biosynthesis was severely impaired by the lack of phaB1. CONCLUSIONS: The present results indicate the importance of flux distribution at the acetoacetyl-CoA node in R. eutropha for the biosynthesis of the PHA copolyesters with regulated composition from structurally unrelated compounds.

DOI： 10.1186/s12934-019-1197-7

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Ralstonia eutropha H16 contains both NADH- and NADPH-dependent reduction activities to acetoacetyl-CoA, and the NADPH-dependent activity is mediated by PhaB paralogs with (R)-stereospecificity providing (R)-3-hydroxybutyryl (3HB)-CoA monomer for poly((R)-3-hydroxybutyrate) synthesis. In contrast, the gene encoding the NADH-dependent enzyme has not been identified to date. This study focused on the NADH-dependent dehydrogenase with (S)-stereospecificity in R. eutropha, as the (S)-specific reduction of acetoacetyl-CoA potentially competed with the polyester biosynthesis via (R)-3HB-CoA. The NADH-dependent reduction activity decreased to one-half when the gene for H16_A0282 (PaaH1), one of two homologs of clostridial NADH-3HB-CoA dehydrogenase, was deleted. The enzyme responsible for the remaining activity was partially purified and identified as H16_A0602 (Had) belonging to a different family from PaaH1. Gene disruption analysis elucidated that most of the NADH-dependent activity was mediated by PaaH1 and Had. The kinetic analysis using the recombinant enzymes indicated that PaaH1 and Had were both NADH-dependent 3-hydroxyacyl-CoA dehydrogenases with rather broad substrate specificity to 3-oxoacyl-CoAs of C4 to C8. The deletion of had in the R. eutropha strain previously engineered for biosynthesis of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) led to decrease in the C6 composition of the copolyester synthesized from soybean oil, suggesting the role of Had in (S)-specific reduction of 3-oxohexanoyl-CoA with reverse β-oxidation direction. Crotonase ((S)-specific enoyl-CoA hydratase) in R. eutropha H16 was also partially purified and identified as H16_A3307.

DOI： 10.1016/j.jbiosc.2018.08.009

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Random mutagenesis for the hyperthermophilic archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.

DOI： 10.1093/nar/gky1313

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This study aimed to enhance production of polyhydroxybutyrate P(3HB) by a newly engineered strain of Cupriavidus necator NSDG-GG by applying response surface methodology (RSM). From initial experiment of one-factor-at-a-time (OFAT), glucose and urea were found to be the most significant substrates as carbon and nitrogen sources, respectively, for the production of P(3HB). OFAT experiment results showed that the maximum biomass, P(3HB) content, and P(3HB) concentration of 8.95 g/L, 76 wt%, and 6.80 g/L were achieved at 25 g/L glucose and 0.54 g/L urea with an agitation rate of 200 rpm at 30 °C after 48 h. In this study, RSM was applied to optimize the three key variables (glucose concentration, urea concentration, and agitation speed) at a time to obtain optimal conditions in a multivariable system. Fermentation experiments were conducted in shaking flask by cultivation of C. necator NSDG-GG using various glucose concentrations (10-50 g/L), urea concentrations (0.27-0.73 g/L), and agitation speeds (150-250 rpm). The interaction between the variables studied was analyzed by ANOVA analysis. The RSM results indicated that the optimum cultivation conditions were 37.70 g/L glucose, 0.73 g/L urea, and 200 rpm agitation speed. The validation experiments under optimum conditions produced the highest biomass of 12.84 g/L, P(3HB) content of 92.16 wt%, and P(3HB) concentration of 11.83 g/L. RSM was found to be an efficient method in enhancing the production of biomass, P(3HB) content, and P(3HB) concentration by 43, 21, and 74%, respectively.

DOI： 10.1007/s13205-018-1351-7

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The Challenger Deep is the deepest ocean on Earth. The present study investigated microbial community structures and geochemical cycles associated with the trench bottom sediments of the Challenger Deep, the Mariana Trench. The SSU rRNA gene communities found in trench bottom sediments were dominated by the bacteria Chloroflexi (SAR202 and other lineages), Bacteroidetes, Planctomycetes, "Ca. Marinimicrobia" (SAR406), and Gemmatimonadetes and by the archaeal α subgroup of MGI Thaumarchaeota and "Ca. Woesearchaeota" (Deep-sea Hydrothermal Vent Euryarchaeotic Group 6). The SSU rRNA gene sequencing analysis indicated that the dominant populations of the thaumarchaeal α group in hadal water and sediments were similar to each other at the species or genus level. In addition, the co-occurrence of nitrification and denitrification was revealed by the combination of pore water geochemical analyses and quantitative PCR for nitrifiers.

DOI： 10.1264/jsme2.ME17194

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DOI： 10.1038/pj.2017.20

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DOI： 10.3389/fmicb.2014.00100

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DOI： 10.1271/bbb.100730

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2011.0.56.0

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DOI： 10.1016/j.polymdegradstab.2010.02.026

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Language：English   Publisher：THE JAPANESE SOCIETY FOR EXTREMOPHILES  

An open reading frame encoding a chitinase homolog (ChiN1) was found in the genome of extremely halophilic archaeon Halobacterium salinarum NRC-1. ChiN1 is a multidomain enzyme consisting of a chitin-binding domain, a polycystic kidney disease domain and a catalytic domain belonging to glycoside hydrolase family 18. chiN1 gene was successfully expressed in extremely halophilic archaeon Haloarcula japonica TR-1 by employing the promoter sequence of its cell surface glycoprotein gene. A large amount of recombinant ChiN1 was secreted into the culture supernatant. The Ha. japonica-produced ChiN1 was purified and characterized. The optimal pH and temperature of ChiN1 are pH 4.5 and 55°C, respectively. ChiN1 was most active at 1.0 M NaCl and stable over a wide range of NaCl concentration from 1.0 to 4.5 M. This is the first report on a chitinase from extremely halophilic archaeon.

DOI： 10.3118/jjse.9.19

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Other Link： https://jlc.jst.go.jp/DN/JALC/00357868582?from=CiNii

Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2010.0.37.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2010.0.38.0

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1271/bbb.80846

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DOI： 10.1271/bbb.80869

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DOI： 10.1021/bm801391j

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：The Japan Petroleum Institute  

DOI： 10.11523/sekiyu.2009f.0.147.0

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Chitinase J (ChiJ) from alkaliphilic Bacillus sp. strain J813 has a multidomain structure containing a catalytic domain (CatD), a fibronectin type III like domain (FnIIID) and a chitin-binding domain (ChBD). It has been shown that the ChBD binds to an insoluble chitin and enhances its degradation by the CatD. Further binding study of the ChBD was performed with a glutathione-S-transferase fusion protein. This fusion protein showed binding abilities to insoluble chitin and chitosan. Two surface-exposed aromatic residues (Trp541 and Trp542) were found in the tertiary-structure model of ChBD and targeted for mutational analysis. Single and double mutations of the two aromatic residues decreased the chitin- and chitosan-binding abilities. It was revealed that these residues would be important for substrate-binding of the ChBD.

DOI： 10.1093/nass/nrp156

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2009.0.135.0

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1139/V08-047

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DOI： 10.1128/jb.00751-06

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DOI： 10.1074/jbc.M702694200

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DOI： 10.1128/jb.01692-06

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Xylanase J (XynJ) from alkaliphilic Bacillussp. strain 41M-1 is an alkaline xylanase. Structure comparison indicated that there were several specific salt bridges in the catalytic cleft of XynJ compared with neutral xylanases. Mutant enzymes were prepared by substituting several amino acids comprising the salt bridges. Some mutants exhibited acidophilic shift in optimum pH, whereas another showed alkaliphilic shift. These results suggested that the characteristic salt bridges could contribute to the alkaliphily of XynJ.

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Language：English   Publishing type：Research paper (scientific journal)  

beta-1,3-Glucanase F (BglF) from alkaliphilic Nocardiopsis sp. F96 is a single domain enzyme composed of only a catalytic domain. Chimeric BglFs with some carbohydrate-binding domains were constructed and characterized. By connecting the C-terminal additional domain of beta-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain of chitinase J from alkaliphilic Bacillus sp. J813, binding ability and hydrolyzing activity toward insoluble beta-1,3-glucans were both improved.

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Language：Japanese   Publisher：The Japan Petroleum Institute  

DOI： 10.11523/sekiyu.2007f.0.115.0

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1365-2958.2006.05287.x

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DOI： 10.1007/s00792-006-0514-3

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

Alkaliphilic Bacillus sp. strain 41M-1 secretes a xylanase (termed xylanase J) that has an alkaline pH optimum. Xylanase J is a multidomain enzyme and consists of two functional domains: a glycoside hydrolase family 11 catalytic domain and an additional domain of unknown function. Protein engineering study of xylanase J indicated that the functionally unknown domain should be a xylan-binding domain (XBD) belonging to carbohydrate binding module family 36. The XBD bound to insoluble xylan and enhanced hydrolyzing activity of the adjacent catalytic domain. The XBD was successfully displayed on the surface of filamentous phage. Random mutations were introduced into the XBD gene and the repertoire was cloned for display on phage. Sequencing analysis of the xylan-binding activity-deficient mutants revealed that Phe284, Asp286, Asp313, Trp317 and Asp318 might contribute to the xylan-binding activity of XBD. The mutant XBD with amino acid substitution T316I (Thr317 was replaced by Ile) showed higher xylan-binding activity compared to the wild-type XBD. Furthermore, hydrolyzing activity of xylanase J toward insoluble xylan was improved by introducing mutation T316I.

DOI： 10.5458/jag.53.131

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/00279786049?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)  

Xylanase J (XynJ) of alkaliphilic Bacillus sp. strain 41M-1 is a multi-domain enzyme and consists of a glycoside hydrolase (GH) family 11 catalytic domain and an additional xylan-binding domain (XBD) belonging to carbohydrate-binding module (CBM) family 36. Random mutations were introduced into the XBD gene and the repertoire was cloned for display on the surface of filamentous phage. The mutant XBD with amino acid substitution T316I (Thr317 was replaced by Ile) showed higher xylan-binding activity compared to the wild-type XBD. Furthermore, hydrolyzing activity of XynJ toward insoluble xylan was also improved by introducing the mutation T316I.

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1128/jb.187.20.7038-7044.2005

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DOI： 10.1128/aem.71.7.3889-3899.2005

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DOI： 10.1016/j.jbiotec.2004.11.002

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A transducer gene, hjtB, was cloned from genomic DNA of extremely halophilic archaeon Haloarcula japonica. The structural gene consisted of an open reading frame of 984 nucleotides encoding 328 amino acids. RT-PCR analysis revealed that this gene was transcribed in Ha. japonica.

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Language：English   Publisher：THE JAPANESE SOCIETY FOR EXTREMOPHILES  

The ferredoxin (Fd) from Haloarcula japonica possesses a plant-type [2Fe-2S] cluster and is stable at high salt concentrations. Ha. japonica Fd (HjFd) includes an N-terminal additional domain rich in acidic amino acids, as well as a common core domain that contains the Fe-S cluster. The N-terminally HAT-tagged intact HjFd (HAT/HjFd) and spinach/Ha. japonica chimeric Fd (HAT/Sp/HjFd) were prepared and characterized. Escherichia coli-produced HAT/Sp/HjFd and Ha. japonica-produced HAT/HjFd were produced as holoproteins. On the other hand, E. coli-produced HAT/HjFd did not incorporate the Fe-S cluster. These results suggested that the N-terminal domain of HjFd contributed to the polypeptide folding and successive Fe-S cluster incorporation under high salt conditions. Both Ha. japonica-produced HAT/HjFd and E. coli-produced HAT/Sp/HjFd were stable at high salt concentrations (≥1.5 M NaCl), although a reduction in stability was observed at lower concentrations. Lack of the N-terminal domain did not affect the stability of HjFd, indicating that the core domain mainly contributed to the stability of HjFd at high salt concentrations. Solubility of E. coli-produced HAT/Sp/HjFd under high salt conditions was significantly lower than that of Ha. japonica-produced HAT/HjFd. It was revealed that substitution of the N-terminal domain of HjFd to that of spinach Fd injured the solubility of HjFd. Thus, it was concluded that the N-terminal domain of HjFd should perform the essential functions for halophilic adaptation from the folding process through the folded state.

DOI： 10.3118/jjse.4.14

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DOI： 10.1128/jb.186.17.5799-5807.2004

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DOI： 10.1074/jbc.M314187200

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DOI： 10.1128/jb.186.14.4620-4627.2004

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DOI： 10.1128/jb.186.13.4185-4191.2004

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DOI： 10.1128/jb.185.17.5175-5181.2003

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DOI： 10.1074/jbc.M205484200

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DOI： 10.1128/jb.185.1.210-220.2003

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DOI： 10.1074/jbc.M202868200

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DOI： 10.1046/j.1432-1033.2002.03016.x

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DOI： 10.1128/jb.184.13.3689-3698.2002

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DOI： 10.1021/bm0255084

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DOI： 10.1046/j.1432-1033.2002.02849.x

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DOI： 10.1128/JB.184.3.777-784.2002

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DOI： 10.1263/jbb.94.237

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DOI： 10.1074/jbc.M105919200

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DOI： 10.1016/s0162-0134(01)00223-9

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DOI： 10.1107/s0907444900014062

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DOI： 10.1021/bm0056052

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Publishing type：Part of collection (book)   Publisher：Elsevier  

DOI： 10.1016/s0076-6879(01)30385-3

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DOI： 10.1046/j.1432-1327.2001.02034.x

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DOI： 10.1016/S0969-2126(01)00608-6

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DOI： 10.1023/a:1005673218300

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DOI： 10.1139/w99-150

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DOI： 10.1007/s002530051633

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DOI： 10.1023/a:1005572526080

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DOI： 10.1111/j.1574-6968.1999.tb13660.x

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DOI： 10.1016/s0141-8130(99)00017-3

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DOI： 10.1016/s0141-8130(99)00018-5

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DOI： 10.1271/bbb.63.870

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DOI： 10.1111/j.1574-6968.1999.tb13356.x

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DOI： 10.1111/j.1574-6968.1998.tb13089.x

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DOI： 10.1007/s002530051185

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DOI： 10.1023/a:1018436426032

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DOI： 10.1007/s002530050898

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DOI： 10.1007/s002530050697

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DOI： 10.1007/s002530050648

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DOI： 10.1246/bcsj.69.515

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DOI： 10.1007/BF00186963

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DOI： 10.1016/s0957-4166(00)80486-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa Healthcare  

DOI： 10.3109/10242429408992133

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DOI： 10.3109/10242429408992133

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DOI： 10.1007/BF00242942

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DOI： 10.1111/j.1749-6632.1992.tb35653.x

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Publisher：工学図書  

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Language：Japanese  

CiNii Books

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Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)  

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Publisher：工学図書  

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Publisher：エヌ・ティー・エス  

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DOI： 10.3118/jjse.4.61

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Publisher：シーエムシー出版  

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Publisher：エヌ・ティー・エス  

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Language：Japanese   Publisher：学会出版センタ-  

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Language：Japanese   Publisher：The Crystallographic Society of Japan  

DOI： 10.5940/jcrsj.40.Supplement_108

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Language：Japanese   Publisher：包装食品技術協会  

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Publisher：1996 International Symposium on Bacterial Polyhydroxyalkanoates  

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Language：Japanese   Publisher：日本生物工学会  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=299634

Language：Japanese   Publisher：日本生物工学会  

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Other Link： http://dl.ndl.go.jp/info:ndljp/pid/10512329

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Applicant：国立大学法人東京工業大学, 株式会社カネカ

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Publication no：WO2017-051896  Date published：2017.3

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Applicant：国立大学法人東京工業大学

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Applicant：国立大学法人東京工業大学

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Patent/Registration no：特許第3771475号  Date registered：2006.2 

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Applicant：独立行政法人科学技術振興機構, 独立行政法人理化学研究所

Application no：特願平10-268791  Date applied：1998.9

Announcement no：特開2000-135083  Date announced：2000.5

Patent/Registration no：特許第4258577号  Date registered：2009.2 

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Applicant：株式会社カネカ, 独立行政法人理化学研究所

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Patent/Registration no：特許第4177912号  Date registered：2008.8 

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Applicant：理化学研究所

Application no：特願平9-199979  Date applied：1997.7

Announcement no：特開平10-108682  Date announced：1998.4

Patent/Registration no：特許第3062459号  Date registered：2000.4 

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