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Novel linear and branched polyamines in the extremely thermophilic eubacteria Thermoleophilum, Bacillus and Hydrogenobacter.

ABSTRACT: Novel tertiary branched tetra-amines, quaternary branched penta-amines, linear penta-amines and linear hexa-amines were distributed as the major polyamines in six obligately extremely thermophilic eubacteria belonging to Thermoleophilum, Bacillus or Hydrogenobacter. The major polyamine of Thermoleophilum album and Thermoleophilum minutum was identified as a quaternary branched penta-amine, 4,4-bis(3-aminopropyl)-1,8-diamino-4-azaoctane (NH2[CH2]3N+([CH2]3NH2)2[CH2]4NH2) by h.p.l.c., t.l.c. and g.c.-m.s. Hydrogenobacter thermophilus and Hydrogenobacter halophilus contained another quaternary branched penta-amine, 4,4-bis(3-aminopropyl)-1,7-diamino-4-azaheptane (NH2[CH2]3N([CH2]3NH2)2[CH2]3NH2) as the major polyamine, and tertiary branched tetra-amines (4-(3-aminopropyl)-1,7-diamino-4-azaheptane (NH2[CH2]3N([CH2]3NH2)[CH2]3NH2), 4-(3-aminopropyl)-1,8-diamino-4-azaoctane (NH2[CH2]3N([CH2]3NH2)[CH2]4NH2)) and 4,4-bis(3-aminopropyl)-1,8-diamino-4-azaoctane were confirmed as minor components. Bacillus schlegelii contained a branched tetra-amine, 4-(3-aminopropyl)-1,8-diamino-4-azaoctane, a branched penta-amine, 4,4-bis(3-aminopropyl)-1,8-diamino-4-azaoctane, a linear penta-amine, 1,16-diamino-4,8,13-triazahexadecane (NH2[CH2]3NH[CH2]3NH[CH2]4NH[CH2]3NH2) and linear hexa-amine(s), 1,20-diamino-4,8,12,17-tetra-azaeicosane (NH2[CH2]3NH[CH2]3NH[CH2]3NH[CH2]4NH[CH2]3NH2 ) and/or 1,20-diamino-4,8,13,17-tetra-azaeicosane (NH2[CH2]3NH[CH2]3NH[CH2]4NH[CH2]3NH[CH2]3NH2 ).

SUBMITTER: Hamana K

PROVIDER: S-EPMC1132601 | biostudies-other | 1992 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Most studies on the thermophilic CBP concentrate on co-cultivation of the thermophilic cellulolytic bacterium Clostridium thermocellum with non-cellulolytic thermophilic anaerobes at temperatures of 55°C-60°C.ResultsWe have specifically screened for cellulolytic bacteria growing at temperatures >70°C to enable direct conversion of lignocellulosic materials into ethanol. Seven new strains of extremely thermophilic anaerobic cellulolytic bacteria of the genus Caldicellulosiruptor and eight new strains of extremely thermophilic xylanolytic/saccharolytic bacteria of the genus Thermoanaerobacter isolated from environmental samples exhibited fast growth at 72°C, extensive lignocellulose degradation and high yield ethanol production on cellulose and pretreated lignocellulosic biomass. Monocultures of Caldicellulosiruptor strains degraded up to 89-97% of the cellulose and hemicellulose polymers in pretreated biomass and produced up to 72 mM ethanol on cellulose without addition of exogenous enzymes. In dual co-cultures of Caldicellulosiruptor strains with Thermoanaerobacter strains the ethanol concentrations rose 2- to 8.2-fold compared to cellulolytic monocultures. A co-culture of Caldicellulosiruptor DIB 087C and Thermoanaerobacter DIB 097X was particularly effective in the conversion of cellulose to ethanol, ethanol comprising 34.8 mol% of the total organic products. In contrast, a co-culture of Caldicellulosiruptor saccharolyticus DSM 8903 and Thermoanaerobacter mathranii subsp. mathranii DSM 11426 produced only low amounts of ethanol.ConclusionsThe newly discovered Caldicellulosiruptor sp. strain DIB 004C was capable of producing unexpectedly large amounts of ethanol from lignocellulose in fermentors. The established co-cultures of new Caldicellulosiruptor strains with new Thermoanaerobacter strains underline the importance of using specific strain combinations for high ethanol yields. These co-cultures provide an efficient CBP pathway for ethanol production and represent an ideal starting point for development of a highly integrated commercial ethanol production process.

Project description:Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N(4)-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, an isomer of N(4)-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N(4)-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N(4)-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N(4)-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N(4)-bis(aminopropyl)spermidine from spermidine via N(4)-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N(4)-aminopropylspermidine and N(4)-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments.

Project description:Genomes of extremely thermophilic Caldicellulosiruptor species encode novel cellulose binding proteins, called t?pirins, located proximate to the type IV pilus locus. The C-terminal domain of Caldicellulosiruptor kronotskyensis t?pirin 0844 (Calkro_0844) is structurally unique and has a cellulose binding affinity akin to that seen with family 3 carbohydrate binding modules (CBM3s). Here, full-length and C-terminal versions of t?pirins from Caldicellulosiruptor bescii (Athe_1870), Caldicellulosiruptor hydrothermalis (Calhy_0908), Caldicellulosiruptor kristjanssonii (Calkr_0826), and Caldicellulosiruptor naganoensis (NA10_0869) were produced recombinantly in Escherichia coli and compared to Calkro_0844. All five t?pirins bound to microcrystalline cellulose, switchgrass, poplar, and filter paper but not to xylan. Densitometry analysis of bound protein fractions visualized by SDS-PAGE revealed that Calhy_0908 and Calkr_0826 (from weakly cellulolytic species) associated with the cellulose substrates to a greater extent than Athe_1870, Calkro_0844, and NA10_0869 (from strongly cellulolytic species). Perhaps this relates to their specific needs to capture glucans released from lignocellulose by cellulases produced in Caldicellulosiruptor communities. Calkro_0844 and NA10_0869 share a higher degree of amino acid sequence identity (>80% identity) with each other than either does with Athe_1870 (?50%). The levels of amino acid sequence identity of Calhy_0908 and Calkr_0826 to Calkro_0844 were only 16% and 36%, respectively, although the three-dimensional structures of their C-terminal binding regions were closely related. Unlike the parent strain, C. bescii mutants lacking the t?pirin genes did not bind to cellulose following short-term incubation, suggesting a role in cell association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, t?pirins likely help scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species.IMPORTANCE The mechanisms by which microorganisms attach to and degrade lignocellulose are important to understand if effective approaches for conversion of plant biomass into fuels and chemicals are to be developed. Caldicellulosiruptor species grow on carbohydrates from lignocellulose at elevated temperatures and have biotechnological significance for that reason. Novel cellulose binding proteins, called t?pirins, are involved in the way that Caldicellulosiruptor species interact with microcrystalline cellulose, and additional information about the diversity of these proteins across the genus, including binding affinity and three-dimensional structural comparisons, is provided here.

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Novel linear and branched polyamines in the extremely thermophilic eubacteria Thermoleophilum, Bacillus and Hydrogenobacter.

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