Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance.

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.

Microaerobic insights into production of polyhydroxyalkanoates containing 3-hydroxyhexanoate via native reverse ß-oxidation from glucose in Ralstonia eutropha H16.

BACKGROUND: Ralstonia eutropha H16, a facultative chemolitoautotroph, is an important workhorse for bioindustrial production of useful compounds such as polyhydroxyalkanoates (PHAs). Despite the extensive studies to date, some of its physiological properties remain not fully understood. RESULTS: This study demonstrated that the knallgas bacterium exhibited altered PHA production behaviors under slow-shaking condition, as compared to its usual aerobic condition. One of them was a notable increase in PHA accumulation, ranging from 3.0 to 4.5-fold in the mutants lacking of at least two NADPH-acetoacetyl-CoA reductases (PhaB1, PhaB3 and/or phaB2) when compared to their respective aerobic counterpart, suggesting the probable existence of (R)-3HB-CoA-providing route(s) independent on PhaBs. Interestingly, PHA production was still considerably high even with an excess nitrogen source under this regime. The present study further uncovered the conditional activation of native reverse ß-oxidation (rBOX) allowing formation of (R)-3HHx-CoA, a crucial precursor for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)], solely from glucose. This native rBOX led to the natural incorporation of 3.9 mol% 3HHx in a triple phaB-deleted mutant (∆phaB1∆phaB1∆phaB2-C2). Gene deletion experiments elucidated that the native rBOX was mediated by previously characterized (S)-3HB-CoA dehydrogenases (PaaH1/Had), ß-ketothiolase (BktB), (R)-2-enoyl-CoA hydratase (PhaJ4a), and unknown crotonase(s) and reductase(s) for crotonyl-CoA to butyryl-CoA conversion prior to elongation. The introduction of heterologous enzymes, crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) along with (R)-2-enoyl-CoA hydratase (PhaJ) aided the native rBOX, resulting in remarkably high 3HHx composition (up to 37.9 mol%) in the polyester chains under the low-aerated condition. CONCLUSION: These findings shed new light on the robust characteristics of Ralstonia eutropha H16 and have the potential for the development of new strategies for practical P(3HB-co-3HHx) copolyesters production from sugars under low-aerated conditions.

Characterization of newly isolated thermotolerant bacterium Cupriavidus sp. CB15 from composting and its ability to produce polyhydroxyalkanoate from glycerol.

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.

(R/S)-lactate/2-hydroxybutyrate dehydrogenases in and biosynthesis of block copolyesters by Ralstonia eutropha.

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 PhaCAR, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaCAR 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 (phaCAR+, ldhACd-hadACd+ 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 phaCAR. 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 PhaCAR 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 identified The engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB.

Characterization of a GlgC homolog from extremely halophilic archaeon Haloarcula japonica.

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.

Random mutagenesis of a hyperthermophilic archaeon identified tRNA modifications associated with cellular hyperthermotolerance.

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.

Modification of acetoacetyl-CoA reduction step in Ralstonia eutropha for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from structurally unrelated compounds.

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 ß-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.

Complete biosynthetic pathway of the C50 carotenoid bacterioruberin from lycopene in the extremely halophilic archaeon Haloarcula japonica.

UNLABELLED: Haloarcula japonica, an extremely halophilic archaeon that requires high concentrations of NaCl for growth, accumulates the C50 carotenoid bacterioruberin (BR). By homology analysis, a gene cluster, including c0507, c0506, and c0505, was found and predicted to be involved in the synthesis of bacterioruberin. To elucidate the function of the encoded enzymes, we constructed Ha. japonica mutants of these genes and analyzed carotenoids produced by the mutants. Our research showed that c0507, c0506, and c0505 encoded a carotenoid 3,4-desaturase (CrtD), a bifunctional lycopene elongase and 1,2-hydratase (LyeJ), and a C50 carotenoid 2",3"-hydratase (CruF), respectively. The above three carotenoid biosynthetic enzymes catalyze the reactions that convert lycopene to bacterioruberin in Ha. japonica. This is the first identification of functional CrtD and CruF in archaea and elucidation of the complete biosynthetic pathway of bacterioruberin from lycopene. IMPORTANCE: Haloarcula japonica, an extremely halophilic archaeon, accumulates the C50 carotenoid bacterioruberin (BR). In this study, we have identified three BR biosynthetic enzymes and have elucidated their functions. Among them, two enzymes were found in an archaeon for the first time. Our results revealed the biosynthetic pathway responsible for production of BR in Ha. japonica and provide a basis for investigating carotenoid biosynthetic pathways in other extremely halophilic archaea. Elucidation of the carotenoid biosynthetic pathway in Ha. japonica may also prove useful for producing the C50 carotenoid BR efficiently by employing genetically modified haloarchaeal strains.

Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha.

Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)], a flexible and practical kind of polyhydroxyalkanoates, is generally produced from plant oils and fatty acids by several wild and recombinant bacteria. This study established an improved artificial pathway for the biosynthesis of P(3HB-co-3HHx) with high 3HHx composition from structurally unrelated fructose in Ralstonia eutropha. Depression of (R)-specific reduction of acetoacetyl-CoA by the deletion of phaB1 was an effective modification for formation of the C6-monomer unit from fructose driven by crotonyl-CoA carboxylase/reductase (Ccr). Co-overexpression of phaJ4a, which encodes medium-chain-length (R)-enoyl-CoA hydratase, with ccr promoted the incorporation of both 3HB and 3HHx units. Further introduction of emdMm, a synthetic gene encoding ethylmalonyl-CoA decarboxylase derived from mouse, was remarkably effective for P(3HB-co-3HHx) biosynthesis, probably by converting ethylmalonyl-CoA generated by the reductive carboxylase activity of Ccr back into butyryl-CoA. A high cellular content of P(3HB-co-3HHx) composed of 22mol% 3HHx could be produced from fructose by the engineered strain of R. eutropha with ΔphaB1 genotype expressing ccr, phaJ4a, and emd.

Compositional regulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by replacement of granule-associated protein in Ralstonia eutropha.

BACKGROUND: Phasin (PhaP), a kind of polyhydroxyalkanoate (PHA) granule-associated proteins, has a role in controlling the properties of PHA granules surface, and is thought to have influence on PHA biosynthesis in PHA-producing bacteria. This study focused on the phaP1(Re) locus in Ralstonia eutropha as a site of chromosomal modification for production of flexible poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. RESULTS: Considering the high expression level of phaP1(Re), phaJ(Ac) [encoding (R)-specific enoyl-CoA hydratase from Aeromonas caviae] was inserted into the downstream of phaP1(Re) on chromosome 1 of R. eutropha strain NSDG harboring phaC(NSDG) (encoding PHA synthase with broad substrate specificity). The constructed strain efficiently accumulated P(3HB-co-3HHx) on soybean oil with higher 3HHx composition when compared to the previous strain having phaJ(Ac) within pha operon. Insertion of the second phaC(NSDG) along with phaJ(Ac) at the phaP1(Re) locus led to incorporation of much larger 3HHx fraction into PHA chains, although the molecular weight was markedly reduced. The R. eutropha strains were further engineered by replacing phaP1(Re) with phaP(Ac) (encoding phasin from A. caviae) on the chromosome. Interestingly, the phasin replacement increased 3HHx composition in the soybean oil-based PHA with keeping high cellular contents, nevertheless no modification was conducted in the metabolic pathways. Kinetic and Western blot analyses of PHA synthase using cellular insoluble fractions strongly suggested that the phasin replacement not only enhanced activity of PHA synthase from A. caviae but also increased affinity especially to longer (R)-3HHx-CoA. It was supposed that the increased affinity of PHA synthase to (R)-3HHx-CoA was responsible for the higher 3HHx composition in the copolyester. CONCLUSIONS: The downstream of phaP1(Re) was a useful site for integration of genes to be overexpressed during PHA accumulation in R. eutropha. The results also clarified that polymerization properties of PHA synthase was affected by the kind of phasin co-existed on the surface of PHA granules, leading to altered composition of the resulting P(3HB-co-3HHx). The phasin replacement is a novel engineering strategy for regulation of composition of PHA copolyesters.

Genetic examination and mass balance analysis of pyruvate/amino acid oxidation pathways in the hyperthermophilic archaeon Thermococcus kodakarensis.

The present study investigated the simultaneous oxidation of pyruvate and amino acids during H2-evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ΔhyhBGSL strain) and the parent strain indicated that NADPH generated via H2 uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ΔporDAB and ΔvorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H2 yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH3, suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.

Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the ß subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2ß2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.

Characterization and gene deletion analysis of four homologues of group 3 pyridine nucleotide disulfide oxidoreductases from Thermococcus kodakarensis.

Enzymatic characterization of the four group 3 pyridine nucleotide disulfide oxidoreductase (PNDOR) homologues TK1299, TK0304, TK0828, and TK1481 from Thermococcus kodakarensis was performed, with a focus on their CoA-dependent NAD(P)H: elemental sulfur (S(0)) oxidoreductase (NSR) and NAD(P)H oxidase (NOX) activities. TK1299 exhibited NSR activity with a preference for NADPH and showed strict CoA-dependency similar to that of the Pyrococcus furiosus homologue PF1186. During the assays, the non-enzymatic formation of H2S from S(0) and free CoA-SH was observed, and the addition of enzyme and NADPH enhanced H2S evolution. A catalytic cycle of TK1299 was proposed suggesting that CoA-SH acted to solubilize S(0) by forming CoA persulfides, followed by reduction of an enzyme-S-S-CoA intermediate produced after both enzymatic and non-enzymatic evolution of H2S from the CoA persulfide, with NADPH as an electron donor. TK1481 showed NSR activity independently of CoA-SH, implying a direct reaction with S(0). TK1299, TK1481, and TK0304 exhibited high NOX activity, and the NADH-dependent activities were inhibited by the addition of free CoA-SH. Multiple disruptions of the four group 3 PNDOR homologues in T. kodakarensis demonstrated that none of these homologues were essential for S(0)-dependent growth. Many disruptants grew better than the parent strain, but a few multiple disruptants showed decreased growth properties after aerobic inoculation into a pyruvate-containing medium without S(0), suggesting the complicated participation of these group 3 PNDORs in sensitivity/resistance to dissolved oxygen when S(0) was absent.

Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates.

Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK Ec or glpK Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.

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 Co²âº-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 Co²âº 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 AM1C(Ac) in which the original PHA synthase gene phaC(Me) had been replaced by phaC(Ac), 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 AM1C(Ac) were higher than those of PHA in the wild-type strain. The triple deletion of three PHA depolymerase genes in M. extorquens AM1C(Ac) showed no significant effects on growth and PHA biosynthesis properties. Overexpression of the genes encoding ß-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.

Gas fermentation combined with water electrolysis for production of polyhydroxyalkanoate copolymer from carbon dioxide by engineered Ralstonia eutropha.

A recycled-gas closed-circuit culture system was developed for safe autotrophic cultivation of a hydrogen-oxidizing, polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha, using a non-combustible gas mixture with low-concentration of H2 supplied by water electrolysis. Automated feedback regulation of gas flow enabled input of H2, CO2, and O2 well balanced with the cellular demands, leading to constant gas composition throughout the cultivation. The engineered strain of R. eutropha produced 1.71 g/L of poly(3-hydroxybutyrate-co-12.5 mol% 3-hydroxyhexanoate) on a gas mixture of H2/CO2/O2/N2 = 4:12:7:77 vol% with a 69.2 wt% cellular content. Overexpression of can encoding cytosolic carbonic anhydrase increased the 3HHx fraction up to 19.6 mol%. The yields of biomass and PHA on input H2 were determined to be 72.9 % and 63.1 %, corresponding to 51.0 % and 44.2 % yield on electricity, respectively. The equivalent solar-to-biomass/PHA efficiencies were estimated to be 2.1-3.8 %, highlighting the high energy conversion capability of R. eutropha.

Thermostable alcohol dehydrogenase from Thermococcus kodakarensis KOD1 for enantioselective bioconversion of aromatic secondary alcohols.

A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent K(m) values for the cofactors NAD(P)(+) and NADPH were similar within a range of 66 to 127 µM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O-20% 2-propanol and H2O-50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.

Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly (3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses.

BACKGROUND: Ralstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. To obtain further knowledge of PHA biosynthesis, this study performed a quantitative transcriptome analysis based on deep sequencing of the complementary DNA generated from the RNA (RNA-seq) of R. eutropha H16. RESULTS: Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined using an Illumina high-throughput sequencer. The RNA-seq results indicated the induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in the growth phase; and the repression trends of genes involved in central metabolisms in the PHA production phase. Interestingly, the transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several genes for ß-oxidation were significantly induced in the PHA production phase even when the cells were grown on fructose. Moreover, incorporation of 13C was observed in poly(3-hydroxybutyrate) synthesized by R. eutropha H16 from fructose in the presence of NaH13CO3, and further gene deletion analyses revealed that both of the two ribulose 1,5-bisphosphate carboxylase (Rubiscos) in CBB cycle were actually functional in CO2 fixation under the heterotrophic condition. CONCLUSIONS: The results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha.

Gene analysis, expression, and characterization of an intracellular α-amylase from the extremely halophilic archaeon Haloarcula japonica.

Haloarcula japonica is an extremely halophilic archaeon that requires high concentrations of NaCl to grow. Recently the draft genome sequence of Ha. japonica was determined, and a gene encoding an α-amylase, malA, was identified. The deduced amino acid sequence of MalA, consisting of 663 amino acids, showed homology to α-amylase family enzymes. The sequence did not contain a secretion signal sequence, indicating that the protein is a cytoplasmic enzyme. Transcription of the malA gene was confirmed by reverse transcription (RT)-PCR, and the transcription start site was determined by a 5'-RACE experiment. The malA gene was cloned and expressed in Ha. japonica. The recombinant MalA was purified and characterized. MalA required a high concentration of NaCl for starch-hydrolyzing activity. It showed higher activity on soluble starch, amylose, and amylopectin, and lower activity on glycogen.

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from CO2 via pH-Stat Jar Cultivation of an Engineered Hydrogen-Oxidizing Bacterium Cupriavidus necator.

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.