An Escherichia coli Mutant Having Altered D-Xylose Uptake Activity and Cloning of a Gene for D-Xylose Uptake

An Escherichia coli C600 mutant having an altered D-xylose uptake activity was isolated. The growth rate and D-xylose uptake activity of the mutant grown on the minimal medium with D-xylose at 25°C were much lower than those of the parental strain grown under the same conditions, although the activities of D-xylose-binding proteins and the enzymes involved in D-xylose metabolism were almost the same for the two strains. An uptake study on sugars at the low temperature (25°C) indicated that the mutant was deficient in D-xylose uptake activity. A gene responsible for the D-xylose uptake activity at the low temperature was isolated and cloned onto vector plasmid pBR322. The gene specifically improved the D-xylose uptake activity of the mutant at the low temperature when it was introduced into the mutant cells. Based on these results, it was suggested that another D-xylose transport system other than the D-xylose-binding protein mediated system might be functioning in E. coli cells.     

Cloning,sequencing and overexpression in Escherichia coli of a xylanase gene,xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor

 A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in λZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70^°C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp. Received: 18 April 1995/Received revision: 4 August 1995/Accepted: 22 August 1995     

A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1

A complete gene, xyl10C, encoding a thermophilic endo-1,4-β-xylanase (XYL10C), was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. XYL10C shares highest nucleotide and amino acid sequence identities of 57.3 and 49.7%, respectively, with a putative xylanase from Aspergillus fumigatus Af293 of glycoside hydrolase family 10. A high expression level in P. pastoris (73,400 U ml⁻¹) was achieved in a 3.7–l fermenter. The purified recombinant XYL10C was thermophilic, exhibiting maximum activity at 85°C, which is higher than that reported from any fungal xylanase. The enzyme was also highly thermostable, exhibiting ~100% of the initial activity after incubation at 80°C for 60 min and >87% of activity at 90°C for 10 min. The half lives of XYL10C at 80 and 85°C were approximately 45 and 3 h, respectively. It had two activity peaks at pH 3.0 and 4.5–5.0 (maximum), respectively, and was very acid stable, retaining more than 80% activity after incubation at pH 1.5−6.0 for 1 h. The enzyme was resistant to Co²⁺, Mn²⁺, Cr³⁺ and Ag⁺. The specific activity of XYL10C for oat spelt xylan was 18,831 U mg⁻¹. It also had wide substrate specificity and produced simple products (65.1% xylose, 25.0% xylobiose and 9.9% xylan polymer) from oat spelt xylan.     

Fractionation of sulfur isotopes during dissimilatory reduction of sulfate by a thermophilic gram-negative bacterium at 60 °C

Sulfur isotope (³⁴S/³²S) fractionation during reduction of dissolved sulfate was investigated with a growing batch culture of a thermophilic, gram-negative, sulfate-reducing bacterium (strain MT-96) at 60 °C. The completely oxidizing strain was isolated from geothermally heated sediments of a shallow-water hydrothermal vent in the Mediterranean Sea. The hydrogen sulfide produced in the experiments was enriched in ³²S by approximately 19‰ as compared to sulfate, which indicates that stable isotope discrimination by this thermophile is within the range found previously for mesophilic sulfate-reducing bacteria, and only slightly higher than that observed for the thermophilic gram-positive Desulfotomaculum nigrificans. Received: 1 December 1998 / Accepted: 25 May 1999     

Mesophilic and thermophilic anaerobic digestion of source-sorted organic wastes: effect of ammonia on glucose degradation and methane production

The wet organic fraction of household wastes was digested anaerobically at 37 °C and 55 °C. At both temperatures the volatile solids loading was increased from 1 g l⁻¹ day⁻¹ to 9.65 g l⁻¹ day⁻¹, by reducing the nominal hydraulic retention time from 93 days to 19 days. The volatile solids removal in the reactors at both temperatures for the same loading rates was in a similar range and was still 65% at 19 days hydraulic retention time. Although more biogas was produced in the thermophilic reactor, the energy conservation in methane was slightly lower, because of a lower methane content, compared to the biogas of the mesophilic reactor. The slightly lower amount of energy conserved in the methane of the thermophilic digester was presumably balanced by the hydrogen that escaped into the gas phase and thus was no longer available for methanogenesis. In the thermophilic process, 1.4 g/l ammonia was released, whereas in the mesophilic process only 1 g/l ammonia was generated, presumably from protein degradation. Inhibition studies of methane production and glucose fermentation revealed a K _i (50%) of 3 g/l and 3.7 g/l ammonia (equivalent to 0.22 g/l and 0.28 g/l free NH₃) at 37 °C and a K _i (50%) of 3.5 g/l and 3.4 g/l ammonia (equivalent to 0.69 g/l and 0.68 g/l free NH₃) at 55 °C. This indicated that the thermophilic flora tolerated at least twice as much of free NH₃ than the mesophilic flora and, furthermore, that the thermophilic flora was able to degrade more protein. The apparent ammonia concentrations in the mesophilic and in the thermophilic biowaste reactor were low enough not to inhibit glucose fermentation and methane production of either process significantly, but may have been high enough to inhibit protein degradation. The data indicated either that the mesophilic and thermophilic protein degraders revealed a different sensitivity towards free ammonia or that the mesophilic population contained less versatile protein degraders, leaving more protein undegraded. Received: 26 March 1997 / Received revision: 13 May 1997 / Accepted: 19 May 1997     

A highly thermostable trehalase from the thermophilic bacterium <Emphasis Type="Italic">Rhodothermus marinus</Emphasis>

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K _m of 0.16 mM and a V _max of 81 μmol of trehalose (min)⁻¹ (mg of protein)⁻¹ at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K _i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.     

Effect of ammonia on the anaerobic degradation of protein by a mesophilic and thermophilic biowaste population

The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated. For peptone concentrations from 5 g l⁻¹ to 20 g l⁻¹ the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g. 552 mg l⁻¹ day⁻¹ compared to 320 mg l⁻¹ day⁻¹ at 10 g l⁻¹ peptone. The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH₃/g peptone in the mesophilic cultures. If 0.5–6.5 g l⁻¹ ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed. The thermophilic biowaste cultures were most active if around 1 g ammonia l⁻¹ was present. Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane. Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a K _i (50%) for NH₃ of 92, 95 and 88 mg l⁻¹ at 37 °C and 251, 274 and 297 mg l⁻¹ at 55 °C respectively. This indicated that the thermophilic flora tolerated significantly more NH₃ than the mesophilic flora. In the mesophilic reactor effluent 4.6 × 10⁸ peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 × 10⁷ cfu/ml was observed. Received: 24 April 1998 / Received revision: 26 June 1998 / Accepted: 27 June 1998     

Characterization of a xylanase from a thermophilic strain of <Emphasis Type="Italic">Anoxybacillus pushchinoensis</Emphasis> A8

A facultatively anaerobic, thermophilic, xylanolytic bacterium was isolated from a sample collected from the Diyadin Hot Springs, Turkey. According to morphological, biochemical and molecular identification, this new strain was suggested to be representative of the Anoxybacillus pushchinoensis and it was designated as Anoxybacillus pushchinoensis strain A8. It exhibited 97% similarity to 16S rRNA gene sequence of A. pushchinoensis and 77% DNA homology by DNA-DNA hybridization studies. Q-sepharose and CM-sepharose chromatography was used to purify an extracellular xylanase to >90% purity from this species. The enzyme had a molecular mass of approximately 83 kDa. The enzyme showed optimum activity at pH 6.5 and it was 96% stable over a broad pH range of 6.5–11 for 24 hours. The enzyme had optimum activity at 55°C and it was 100% stable at temperature between 50–60°C up to 24 hours. Kinetic characterization of the enzyme was performed at temperature optima (55°C) and V_max and K _m were found to be 59.88 U/mg protein and 0.909 mg/mL, respectively. Oat spelt xylan but not xylooligosaccharides was degraded by the enzyme and xylose was the only product detected from oat xylan degradation. This suggested that the enzyme was an exo-acting xylanase.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis.

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31° C. Cells are temperature sensitive at 35.5° C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19° C to 25° C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest. Received: 14 October 1998 / Accepted: 15 March 1999     

<Emphasis Type="Italic">Anoxybacillus mongoliensis</Emphasis> sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring,Central Mongolia

A Gram reaction positive, spore-forming, facultative anaerobic bacterium belonging to the Phylum Firmicutes, was isolated from alkaline hot (80°C, pH 9.8) spring Tsenher, Central Mongolia. The cells were rod shaped, feebly motile, peritrichously flagellated. Strain T4T was moderately thermophilic with optimum growth at 60°C. Maximum temperature for growth was between 70 and 75°C; minimum temperature for growth was between 35 and 30°C. Alkalitolerant, optimum pH for growth was 8.0; minimum pH for growth was between 5.0 and 5.5 and maximum was between 10.5 and 10.8. The growth was observed at NaCl concentrations of 0–5% (w/v) with the optimum at 0.2–0.5%. No growth was observed at 6% NaCl (w/v). Aerobically, the strain utilized proteinaceous substrates, organic acids and a range of carbohydrates including glucose, ribose, sucrose and xylose as well. Anaerobically, only glucose and sucrose were utilized. Strain T4^T produced thermostable alkaline subtilisin-like serine proteinase. The G + C content was 44.2 mol % (td). On the basis of 16S rRNA gene sequence similarity strain T4^T was shown to be closely related to the members of the genus Anoxybacillus (family Bacillaceae, class “Bacilli”). DNA-DNA hybridization data revealed that strain T4^T had only 38% relatedness to A. flavithermus and 28% relatedness to A. pushchinoensis. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA relatedness values with validly published species of Anoxybacillus, it is proposed that strain T4^T represents a novel species Anoxybacillus mongoliensis sp. nov., with the type strain T4^T (=DSM 19169 =VKM 2407).     

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50–80°C and pH 6.0–8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA–DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542^T). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.     

Preliminary characterization of a thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41

A thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41 was characterized in this study. The polI was cloned, sequenced and over-expressed in Escherichia coli. The expressed 110 kDa fusion protein of PolI was stable at 70°C for 1 h. Compared with DNA polymerase I of E. coli (TaKaRa), the relative polymerase activity of this PolI was 3.33 ± 0.1 RFU μl⁻¹ at 37°C using fluorescent quantitative analysis. It showed higher polymerase activity than E. coli PolI at higher temperature, with a relative activity of 3.75 ± 0.1 RFU μl⁻¹ at 70°C. The polI sequence analysis and the protein structure prediction indicated that this protein had a high similarly to other PolI from thermophilic micro-organisms. This information is of importance for future study for evolution of the house-keeping gene polI in entomopathogenic bacterium B. sphaericus.     

Nitrile hydratase from a thermophilic Bacillus smithii

A novel thermophilic Bacillus smithii strain SC-J05-1, isolated from a hot spring, had the ability of hydrating nitrile to form amide. The nitrile hydratase was purified to homogeneity from the microbial cells of SC-J05-1 and was characterized. The enzyme was a 130-kDa protein composed of two different subunits (25.3 kDa and 26.8 kDa) and contained cobalt ions. This enzyme had the optimal temperature of 40°C and was stable up to 50°C. The optimal pH was in the alkaline region higher than pH 10. Received 02 September 1997/ Accepted in revised form 06 February 1998     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

High-throughput screening and characterization of xylose-utilizing, ethanol-tolerant thermophilic bacteria for bioethanol production

Aims: To develop a high‐throughput assay for screening xylose‐utilizing and ethanol‐tolerant thermophilic bacteria owing to their abilities to be the promising ethanologens. Methods and Results: Based on alcohol oxidase and peroxidase‐coupled enzymatic reaction, an assay was developed by the formation of the coloured quinonimine to monitor the oxidation of ethanol in the reaction and calculate the concentration of ethanol. This assay was performed in 96‐well microtitre plate in a high‐throughput and had a well‐linear detection range of ethanol from 0 up to 2·5 g l^?1 with high accuracy. The assay was then verified by screening soil samples from hot spring for xylose‐utilizing and ethanol production at 60°C. Three isolates LM14‐1, LM14‐5 and LM18‐4 with 3–5% (v/v) ethanol tolerance and around 0·29–0·38 g g^?1 ethanol yield from xylose were obtained. Phylogenetic and phenotypic analysis showed that the isolates clustered with members of the genus Bacillus or Geobacillus subgroup. Conclusions: The developed double enzyme‐coupled, high‐throughput screening system is effective to screen and isolate xylose‐utilizing, ethanol‐producing thermophilic bacteria for bioethanol production at the elevated temperature. Significance and Impact of the Study: Our research presented a novel high‐throughput method to screen thermophilic bacteria for producing ethanol from xylose. This screening method is also very useful to screen all kinds of ethanologens either from natural habitats or from mutant libraries, to improve bioethanol production from lignocellulosic feedstocks.     

Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol

The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.     

Nutritional Requirements of <Emphasis Type="Italic">Allisonella histaminiformans,</Emphasis> a Ruminal Bacterium that Decarboxylates Histidine and Produces Histamine

A xylose ABC (ATP-binding cassette) transport operon, xylFGH, was cloned from Thermoanaerobacter ethanolicus, a thermophilic ethanol-producing eubacterium. The cistrons code for a periplasmic D-xylose-binding protein (XylF, partial sequence of 250 amino acids), ATP-binding protein (XylG, 505 amino acids), and integral membrane protein (XylH, 388 amino acids). These results, together with previous work, indicate that duplicate copies of both xylF and xylH are present in the T. ethanolicus chromosome, suggesting ancient gene duplication or lateral gene transfer events. XylG resembles other eubacterial monosaccharide ABC-ATPases in that its two nucleotide-binding domains (NBDs) are highly homologous, yet significantly different with respect to putative catalytic residues. Unlike most other integral membrane ABC transport proteins, XylH apparently contains 11 or 12 transmembrane segments (TMS) and is similar to a small group of ABC permeases that defy the 2 × 6 helix paradigm. This is the first report of a monosaccharide ABC transport operon in a thermophilic anaerobic eubacterium.     

Kinetic comparisons of mesophilic and thermophilic aerobic biomass

Kinetic parameters describing growth and decay of mesophilic (30°C) and thermophilic (55°C) aerobic biomass were determined in continuous and batch experiments by using oxygen uptake rate measurements. Biomass was cultivated on a single soluble substrate (acetate) in a mineral medium. The intrinsic maximum growth rate (μ _max) at 55°C was 0.71±0.09 h⁻¹, which is 1.5 times higher than the μ _max at 30°C (0.48±0.11 h⁻¹). The biomass decay rates increased from 0.004 h⁻¹ at 30°C to 0.017 h⁻¹ at 55°C. Monod constants were very low for both types of biomass: 9±2 mg chemical oxygen demand (COD) l⁻¹at 30°C and 3±2 mg COD l⁻¹at 55°C. Theoretical biomass yields were similar at 30 and 55°C: 0.5 g biomass COD (g acetate COD)⁻¹. The observed biomass yields decreased under both temperature conditions as a function of the cell residence time. Under thermophilic conditions, this effect was more pronounced due to the higher decay rates, resulting in lower biomass production at 55°C compared to 30°C. Electronic Publication     

Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis

Candida tropicalis, a strain isolated from the sludge of a factory manufacturing xylose, produced a high xylitol concentration of 131 g/l from 150 g/l xylose at 45 h in a flask. Above 150 g/l xylose, however, volumetric xylitol production rates decreased because of a lag period in cell growth. In fed-batch culture, the volumetric production rate and xylitol yield from xylose varied substantially with the controlled xylose concentration and were maximum at a controlled xylose concentration of 60 g/l. To increase the xylitol yield from xylose, feeding experiments using different ratios of xylose and glucose were carried out in a fermentor. The maximum xylitol yield from 300 g/l xylose was 91% at a glucose/xylose feeding ratio of 15%, while the maximum volumetric production rate of xylitol was 3.98 g l⁻¹ h⁻¹ at a glucose/xylose feeding ratio of 20%. Xylitol production was found to decrease markedly as its concentration rose above 250 g/l. In order to accumulate xylitol to 250 g/l, 270 g/l xylose was added in total, at a glucose/xylose feeding ratio of 15%. Under these conditions, a final xylitol production of 251 g/l, which corresponded to a yield of 93%, was obtained from 270 g/l xylose in 55 h. Received: 20 April 1998 / Received revision: 29 May 1998 / Accepted: 19 June 1998