Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2

(S)- and (R)-3-hydroxybutyrate (3HB) are precursors to synthesize the biodegradable plastics polyhydroxyalkanoates (PHAs) and many fine chemicals. To date, however, their production has been restricted to petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economical feasibility. With the ability to fix CO₂ photosynthetically, cyanobacteria have attracted increasing interest as a biosynthesis platform to produce fuels and chemicals from alternative renewable resources. To this end, synthesis metabolic pathways have been constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803 to photosynthetically produce (S)- and (R)-3HB directly from CO₂. Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing pathway by deleting slr1829 and slr1830 (encoding PHB polymerase) from the Synechocystis genome further promoted the 3HB production. Up to 533.4 mg/L 3HB has been produced after photosynthetic cultivation of the engineered cyanobacterium Synechocystis TABd for 21 days. Further analysis indicated that the phosphate consumption during the photoautrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. For the first time, the study has demonstrated the feasibility of photosynthetic production of (S)- and (R)-3HB directly from sunlight and CO₂.     

Biochemical and histomorphological findings in Swiss Wistar rats treated with potential boron-containing therapeutic - K2[B3O3F4OH]

BackgroundBoron and boron containing compounds are known for their biological and protective roles being non-toxic and non-mutagenic in low concentrations. Male rats were exposed to halogenated boroxine (HB), dipotassium-trioxohydroxytetrafluorotriborate K₂[B₃O₃F₄OH], a potential new boron-containing therapeutic, aiming to determine concentrations with no adverse effects on selected serum biochemical parameters and histomorphological features.MethodsHB was prepared by reacting potassium hydrofluoride (KHF₂) with boric acid in molar ratios 2:3 at room temperature and its primary structure contains 4 fluorine atoms substituted in 6-membered ring. In concentrations of 10, 25, 35 and 45 mg/kg, HB was administered intraperitoneally as a single dose. Biochemical parameters were observed 24 and 96 h following the treatment. Effects of HB on biochemical blood parameters were also observed 24 h following continuous nine days application in concentrations of 10 mg/kg intraperitoneally and 50 mg/kg per os. Histomorphological observation of kidneys, liver, spleen, lungs and heart was performed for all treated animals.ResultsAdministration of single high dose of HB (35 mg/kg–45 mg/kg) effected high levels of urea and creatinine, which indicated renal injury that appeared to be temporary. Possible cause of concern is pancreatic injury indicated by elevated levels of serum amylase in the groups of animals that received the highest dosages of the substance. Histopathological examination of selected tissues revealed mild to moderate lesions in the kidneys and livers associated with administration of HB.ConclusionObservation of biochemical serum parameters or histopathology of examined tissues revealed no adverse effects of HB either after the administration of single dose lower than 35 mg/kg or following repeated administration at 10 mg/kg. These dosages should be further considered for potential therapeutic applications.     

Pilot scale production of poly(3-hydroxybutyrate-co-3-hydroxy-valerate) by fed-batch culture of recombinantEscherichia coli

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB/V)], by fed-batch culture of recombinantEscherichia coli harboring a plasmid containing theAlcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes, was examined in two pilot-scale fermentors with air supply only. In a 30 L fermentor having aK _La value of 0.11 s⁻¹, the final P(3HB/V) concentration and the P(3HB/V) content obtained were 29.6 g/L and 70.1 wt%, respectively, giving a productivity of 1.37 g P(3HB/V)/L-h. In a 300 L fermentor having aK _La of 0.03 s⁻¹, the P(3HB/V) concentration and the P(3HB/V) content were 20.4 g/L and 69 wt%, respectively, giving a productivity of 1.06 g P(3HB/V)/L-h. These results suggest that economical production of P(3HB/V) is possible by fed-batch culture of recombinantE. coli in a large-scale fermentor having lowK _La value.     

3-Carboxamido-5-aryl-isoxazoles as new CB2 agonists for the treatment of colitis

Recent investigations showed that anandamide, the main endogenous ligand of CB₁ and CB₂ cannabinoid receptors, possesses analgesic, antidepressant and anti-inflammatory effects. In the perspective to treat inflammatory bowel disease (IBD), our approach was to develop new selective CB₂ receptor agonists without psychotropic side effects associated to CB₁ receptors. In this purpose, a new series of 3-carboxamido-5-aryl-isoxazoles, never described previously as CB₂ receptor agonists, was designed, synthesized and evaluated for their biological activity. The pharmacological results have identified great selective CB₂ agonists with in vivo anti-inflammatory activity in a DSS-induced acute colitis mouse model.     

Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters

Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1_Ps) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1_Ps that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436–2442). In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1_Ps was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1_Ps mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired mutants.     

Biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate from unrelated carbon source by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (Pct _Cp ) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Using this in vivo PLA biosynthesis system, we presently report the biosynthesis of PHAs containing 2-hydroxybutyrate (2HB) monomer by direct fermentation of a metabolically engineered E. coli strain. The recombinant E. coli ldhA mutant XLdh strain expressing PhaC1 _Ps6-19 and Pct _Cp was developed and cultured in a chemically defined medium containing 20 g/L of glucose and varying concentrations of 2HB and 3HB. PHAs consisting of 2HB, 3HB, and a small fraction of lactate were synthesized. Their monomer compositions were dependent on the concentrations of 2HB and 3HB added to the culture medium. Even though the ldhA gene was completely deleted in the chromosome of E. coli, up to 6 mol% of lactate was found to be incorporated into the polymer depending on the culture condition. In order to synthesize PHAs containing 2HB monomer without feeding 2HB into the culture medium, a heterologous metabolic pathway for the generation of 2HB from glucose was constructed via the citramalate pathway, in which 2-ketobutyrate is synthesized directly from pyruvate and acetyl-CoA. Introduction of the Lactococcus lactis subsp. lactis Il1403 2HB dehydrogenase gene (panE) into E. coli allowed in vivo conversion of 2-ketobutyrate to 2HB. The metabolically engineered E. coli XLdh strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene successfully produced PHAs consisting of 2HB, 3HB, and a small fraction of lactate by varying the 3HB concentration in the culture medium. As the 3HB concentration in the medium increased the 3HB monomer fraction in the polymer, the polymer content increased. When Ralstonia eutropha phaAB genes were additionally expressed in this recombinant E. coli XLdh strain, P(2HB-co-3HB-co-LA) having small amounts of 2HB and LA monomers could also be produced from glucose as a sole carbon source. The metabolic engineering strategy reported here should be useful for the production of PHAs containing 2HB monomer.     

Complete Dehydrogenation of N2H4BH3 over Noble‐Metal‐Free Ni0.5Fe0.5–CeOx/MIL‐101 with High Activity and 100% H2 Selectivity

Efficient and selective dehydrogenation of hydrazine borane (HB), a novel hydrogen storage material with very high hydrogen content (HB, 15.4 wt%), is a key challenge for a fuel‐cell‐based hydrogen economy. However, even using the noble metal catalysts for HB decomposition, the activities are still far from satisfying, to say nothing of non‐noble‐metal‐containing catalysts. In response, as a proof‐of‐concept experiment, herein, noble‐metal‐free NiFe–CeO_x nanoparticles are successfully immobilized on an MIL‐101 support without surfactant by a simple liquid impregnation method. Unexpectedly, the resultant Ni_0.5Fe_0.5–CeO_x/MIL‐101 catalyst shows good performance, including 100% H₂ selectivity, 100% conversion, and record catalytic activity (351.3 h^?1) for hydrogen generation at mild temperature, which is even better than most of the noble metal heterogeneous catalysts and might be attributed to the good dispersion and uniform particle size of the Ni_0.5Fe_0.5–CeO_x nanoparticles due to steric restrictions effect of the MIL‐101 support. Additionally, extending MIL‐101 to some other important kinds of metal–organic framework (MOF) structures, the resultant NiFe–CeO_x/MOF catalysts all show good catalytic activity toward HB decomposition, showing the universality of the MOF supported NiFe–CeO_x catalysts.     

Autotaxin–lysophosphatidic acid–LPA3 signaling at the embryo‐epithelial boundary controls decidualization pathways

During pregnancy, up‐regulation of heparin‐binding (HB‐) EGF and cyclooxygenase‐2 (COX‐2) in the uterine epithelium contributes to decidualization, a series of uterine morphological changes required for placental formation and fetal development. Here, we report a key role for the lipid mediator lysophosphatidic acid (LPA) in decidualization, acting through its G‐protein‐coupled receptor LPA₃ in the uterine epithelium. Knockout of Lpar3 or inhibition of the LPA‐producing enzyme autotaxin (ATX) in pregnant mice leads to HB‐EGF and COX‐2 down‐regulation near embryos and attenuates decidual reactions. Conversely, selective pharmacological activation of LPA₃ induces decidualization via up‐regulation of HB‐EGF and COX‐2. ATX and its substrate lysophosphatidylcholine can be detected in the uterine epithelium and in pre‐implantation‐stage embryos, respectively. Our results indicate that ATX–LPA–LPA₃ signaling at the embryo‐epithelial boundary induces decidualization via the canonical HB‐EGF and COX‐2 pathways.     

Ketone body 3‐hydroxybutyrate mimics calorie restriction via the Nrf2 activator,fumarate, in the retina

Calorie restriction (CR) being the most robust dietary intervention provides various health benefits. D‐3‐hydroxybutyrate (3HB), a major physiological ketone, has been proposed as an important endogenous molecule for CR. To investigate the role of 3HB in CR, we investigated potential shared mechanisms underlying increased retinal 3HB induced by CR and exogenously applied 3HB without CR to protect against ischemic retinal degeneration. The repeated elevation of retinal 3HB, with or without CR, suppressed retinal degeneration. Metabolomic analysis showed that the antioxidant pentose phosphate pathway and its limiting enzyme, glucose‐6‐phosphate dehydrogenase (G6PD), were concomitantly preserved. Importantly, the upregulation of nuclear factor erythroid 2 p45‐related factor 2 (Nrf2), a regulator of G6PD, and elevation of the tricarboxylic acid cycle's Nrf2 activator, fumarate, were also shared. Together, our findings suggest that CR provides retinal antioxidative defense by 3HB through the antioxidant Nrf2 pathway via modification of a tricarboxylic acid cycle intermediate during 3HB metabolism.     

Biosynthesis of copolyesters consisting of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids from 1,3-butanediol or from 3-hydroxybutyrate by Pseudomonas sp. A33

Pseudomonas sp. A33 and other isolates of aerobic bacteria accumulated a complex copolyester containing 3-hydroxybutyric acid (3HB) and various medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from 3-hydroxybutyric acid or from 1,3-butanediol under nitrogen-limitated culture conditions. 3HB contributed to 15.1 mol/100 mol of the constituents of the polyester depending on the strain and on the cultivation conditions. The accumulated polymer was a copolyester of 3HB and 3HA_MCL rather than a blend of poly(3HB) and poly(3HA_MCL) on the basis of multiple evidence. 3-Hydroxyhexadecenoic acid and 3-hydroxyhexadecanoic acid were detected as constituents of polyhydroxyalkanoates, which have hitherto not been described, by¹³C nuclear magnetic resonance or by gas chromatography/mass spectrometric analysis. In total, ten different constituents were detected in the polymer synthesized from 1,3-butanediol by Pseudomonas sp. A33:besides seven saturated (3HB, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydrohexadecanoate) three unsaturated (3-hydroxydodecenoate, 3-hydroxytetradecenoate and 3-hydrohexadecanoate) hydroxyalkanoic acid constituents occured. The polyhydroxyalkanoate synthase of Pseudomonas sp. A33 was cloned, and its substrate specificity was evaluated by heterologous expression in various strains of P. putida, P. oleovorans and Alcaligenes eutrophus.     

Enhanced production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) copolymer with manipulated variables and its properties

Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M _n) of copolymers ranged from 260 × 10³ to 590 × 10³Da, and the polydispersities (M _w/M _n) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T _m), glass transition temperature (T _g), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbA_Re) and NADPH-dependent acetoacetyl-CoA reductase (PhbB_Re) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1 _Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB⁻4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1 _Ps gene only. In addition, recombinant strains of R. eutropha PHB⁻4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB⁻4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units. Received: 12 July 1999 / Received revision: 1 October 1999 / Accepted: 2 October 1999     

A tale of two tails: The importance of unstructured termini in the aggregation pathway of β2‐microglobulin

The identification of intermediate states for folding and aggregation is important from a fundamental standpoint and for the design of novel therapeutic strategies targeted at conformational disorders. Protein human β2‐microglobulin (HB2m) is classically associated with dialysis‐related amyloidosis, but the single point mutant D76N was recently identified as the causative agent of a hereditary systemic amyloidosis affecting visceral organs. Here, we use D76N as a model system to explore the early stage of the aggregation mechanism of HB2m by means of an integrative approach framed on molecular simulations. Discrete molecular dynamics simulations of a structured‐based model predict the existence of two intermediate states populating the folding landscape. The intermediate I₁ features an unstructured C‐terminus, while I₂, which is exclusively populated by the mutant, exhibits two unstructured termini. Docking simulations indicate that I₂ is the key species for aggregation at acidic and physiological pH contributing to rationalize the higher amyloidogenic potential of D76N relative to the wild‐type protein and the ΔN6 variant. The analysis carried out here recapitulates the importance of the DE‐loop in HB2m self‐association at a neutral pH and predicts a leading role of the C‐terminus and the adjacent G‐strand in the dimerization process under acidic conditions. The identification of aggregation hot‐spots is in line with experimental results that support the importance of Phe56, Asp59, Trp60, Phe62, Tyr63, and Tyr66 in HB2m amyloidogenesis. We further predict the involvement of new residues such as Lys94 and Trp95 in the aggregation process.     

Production of a novel copolyester of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids by Pseudomonas sp. 61-3 from sugars

 Pseudomonas sp. 61-3 (isolated from soil) produced a polyester consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA) of C₆, C₈, C₁₀ and C₁₂, when sugars of glucose, fructose and mannose were fed as the sole carbon source. The polyester produced was a blend of homopolymer and copolymer, which could be fractionated with boiling acetone. The acetone-insoluble fraction of the polyester was a homopolymer of 3-hydroxybutyrate units [poly (3HB)], while the acetone-soluble fraction was a copolymer [poly(3HB-co-3HA)] containing both short- and medium-chain-length 3-hydroxyalkanoate units ranging from C₄ to C₁₂:44 mol% 3-hydroxybutyrate, 5 mol% 3-hydroxyhexanoate, 21 mol% 3-hydroxyoctanoate, 25 mol% 3-hydroxydecanoate, 2 mol% 3-hydroxydodecanoate and 3 mol% 3-hydroxy-5-cis-dodecenoate. The copolyester was shown to be a random copolymer of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoate units by analysis of the ¹³C-NMR spectrum. The poly(3HB) homopolymer and poly (3HB-co-3HA) copolymer were produced simultaneously within cells from glucose in the absence of any nitrogen source, which suggests that Pseudomonas sp. 61-3 has two types of polyhydroxy-alkanoate syntheses with different substrate specificities. Received: 9 June 1995/Received last revision: 30 October 1995/Accepted: 6 November 1995     

Accumulation of 2-Aminophenoxazin-3-one-7-Carboxylate during Growth of Pseudomonas putida TW3 on 4-Nitro-Substituted Substrates Requires 4-Hydroxylaminobenzoate Lyase (PnbB)

During growth of Pseudomonas putida strain TW3 on 4-nitrotoluene (4NT) or its metabolite 4-nitrobenzoate (4NB), the culture medium gradually becomes yellow-orange with a λ_max of 446 nm. The compound producing this color has been isolated and identified as a new phenoxazinone, 2-aminophenoxazin-3-one-7-carboxylate (APOC). This compound is formed more rapidly and in greater quantity when 4-amino-3-hydroxybenzoate (4A3HB) is added to growing cultures of strain TW3 and is also formed nonbiologically when 4A3HB is shaken in mineral salts medium but not in distilled water. It is postulated that APOC is formed by the oxidative dimerization of 4A3HB, although 4A3HB has not been reported to be a metabolite of 4NT or a product of 4NB catabolism by strain TW3. Using the cloned pnb structural genes from TW3, we demonstrated that the formation of the phenoxazinone requires 4-hydroxylaminobenzoate lyase (PnbB) activity, which converts 4-hydroxylaminobenzoate (4HAB) to 3,4-dihydroxybenzoate (protocatechuate) and that 4-nitrobenzoate reductase (PnbA) activity, which causes the accumulation of 4HAB from 4NB, does not on its own result in the formation of APOC. This rules out the possibility that 4A3HB is formed abiotically from 4HAB by a Bamberger rearrangement but suggests that PnbB first acts to effect a Bamberger-like rearrangement of 4HAB to 4A3HB followed by the replacement of the 4-amino group by a hydroxyl to form protocatechuate and that the phenoxazinone is produced as a result of some misrouting of the intermediate 4A3HB from its active site.     

Anti--Histaminic potentila of fractions from Hedranthera Barteri [(Hook F.) Pichon] root in laboratory rodents

Hedranthera barteri, anti-inflammatory activity, anti-nociceptive activity, anti-histaminic activityL. (HB) is used in the treatment of painful conditions and oedema amongst its folkloric use. The hexane and ethyl acetate fractions of the root of H. barteri were investigated for anti-nociceptive and antiinflammatory properties and probable mechanism of action. Hot plate, tail flick, formalin-induced oedema and acetic acidinduced writhing tests were employed to investigate the anti-nociceptive activity while the anti-inflammatory activity was investigated using carrageenan-induced paw oedema. Anti-histaminic potential of HB root extracts on the rat peritoneal mast cells (RPMCs) was explored through pectrofluorometric method. The root was screened for its phytochemical components. The HB root contains alkaloids,cardenolides and saponins. HXHBR exhibited higher anti-inflammatory potentials (P <0.001). HXHBR dose-dependently (P <0.01) reduced the histamine release from the rat peritoneal mast cells which is comparable with a standard anti-histaminic drug, ketotifen. These results showed that EAHBR and HXHBR possess anti-nociceptive and anti-inflammatory activities, and suggested its mechanism of action through the inhibition of histamine, an inflammatory mediator, usually released during the early phase of allergic responses and chronic phase of inflammatory pain. Flavonoids, alkaloids and/or saponins present in HB root may be responsible for its anti-nociceptive and anti-inflammatory properties.     

A Pseudomonas strain accumulating polyesters of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids

Summary A citronellol-utilizing bacterium was isolated that accumulated a polyester consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from various carbon sources up to approximately 70% of the cellular dry matter if the cells were cultivated in ammineral salts medium under nitrogen limitation. In octanoate-grown cells, for instance, the polyester consisted of 87.5 mol% 3HB and 12.5 mol% 3-hydroxyoctanoic acid (3HO), whereas it consisted of 10.3 mol% 3HB, 16.7 mol% 3HO and 73.0 mol% 3-hydroxydecanoic acid (3HD) in gluconate-grown cells. However, the results of various experiments indicated that a blend rather than a copolyester was synthesized in the cell. It was the only strain among 45 different recently isolated citronellol-utilizing bacteria that accumulated such a polyester. All other citronellol-utilizing bacteria behaved like Pseudomonas aeruginosa with respect to their polyhydroxyalkanoic acid (PHA) biosynthetic capabilities and accumulated PHA consisting of 3HA_MCL with 3HO and 3HD as the main constituents from octanoate or gluconate, respectively, whereas 3HB was never present. None of 232 different heavy-metal-resistant bacteria was able to accumulate PHA composed of 3HB plus, for example, 3HO. Only 20.3% did not accumulate any PHA at all, 44.8% accumulated PHB from gluconate, and 34.9% behaved like P. aeruginosa. Many bacteria belonging to the latter group were distinguished from the other by rapid growth in nutrient broth and in gluconate mineral salts medium and by their ability to grow in the presence of a high concentration (up to 1.5%, w/v) of octanoate. Correspondence to: A. Steinbüchel     

Bioenergetics at extreme temperature: Thermus thermophilus ba3- and caa3-type cytochrome c oxidases

Seven years into the completion of the genome sequencing projects of the thermophilic bacterium Thermus thermophilus strains HB8 and HB27, many questions remain on its bioenergetic mechanisms. A key fact that is occasionally overlooked is that oxygen has a very limited solubility in water at high temperatures. The HB8 strain is a facultative anaerobe whereas its relative HB27 is strictly aerobic. This has been attributed to the absence of nitrate respiration genes from the HB27 genome that are carried on a mobilizable but highly-unstable plasmid. In T. thermophilus, the nitrate respiration complements the primary aerobic respiration. It is widely known that many organisms encode multiple biochemically-redundant components of the respiratory complexes. In this minireview, the presence of the two cytochrome c oxidases (CcO) in T. thermophilus, the ba₃- and caa₃-types, is outlined along with functional considerations. We argue for the distinct evolutionary histories of these two CcO including their respective genetic and molecular organizations, with the caa₃-oxidase subunits having been initially ‘fused’. Coupled with sequence analysis, the ba₃-oxidase crystal structure has provided evolutionary and functional information; for example, its subunit I is more closely related to archaeal sequences than bacterial and the substrate–enzyme interaction is hydrophobic as the elevated growth temperature weakens the electrostatic interactions common in mesophiles. Discussion on the role of cofactors in intra- and intermolecular electron transfer and proton pumping mechanism is also included. This article is part of a Special Issue entitled: Respiratory Oxidases.     

A new Co(III)-hypocrellin B complex with enhanced photonuclease activity

Hypocrellin B (HB), a naturally occurring photosensitizer, has been extensively and intensively studied as a promising photodynamic therapy (PDT) agent. In this work, a new Co(III) complex [Co₂(HB)(tmp)₄]⁴⁺ (tmp = 3,4,7,8-tetramethyl-1,10-phenanthroline) was designed and synthesized with HB as bridging ligand and tmp as terminal ligand. [Co₂HB(tmp)₄]⁴⁺ exhibits improved water solubility, enhanced absorptivity in the phototherapeutic window, increased binding affinity and DNA photocleavage capability toward dsDNA with respect to HB. The photodynamic activity of [Co₂(HB)(tmp)₄]⁴⁺ stems from its ¹O₂ photosensitization ability, in sharp contrast to [Cu₂(HB)(tmp)₂]²⁺ which relies on superoxide anion radical (O₂^-) and hydroxyl radical (·OH) to photocleave DNA, though the both complexes possess similar electrochemical properties. The remarkable difference between the photodynamic mechanisms of [Co₂(HB)(tmp)₄]⁴⁺ and [Cu₂(HB)(tmp)₂]²⁺ was discussed in detail.