Halomonas alkalitolerans sp. nov., a novel moderately halophilic bacteriun isolated from soda meadow saline soil in Daqing, China.

A moderately halophilic bacterial strain 15-13(T), which was isolated from soda meadow saline soil in Daqing City, Heilongjiang Province, China, was subjected to a polyphasic taxonomic study. The cells of strain 15-13 were found to be Gram-negative, rod-shaped, and motile. The required growth conditions for strain 15-13(T) were: 1-23% NaCl (optimum, 7%), 10-50°C (optimum, 35°C), and pH 7.0-11.0 (optimum, pH 9.5). The predominant cellular fatty acids were C(18:1) ω7c (60.48%) and C(16:0) (13.96%). The DNA G+C content was 67.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain 15-13(T) clustered within a branch comprising species of the genus Halomonas. The closest phylogenetic neighbor of strain 15-13(T) was Halomonas pantelleriensis DSM 9661(T) (98.9% 16S rRNA gene sequence similarity). The level of DNA-DNA relatedness between the novel isolated strain and H pantelleriensis DSM 9661(T) was 33.8%. On the basis of the phenotypic and phylogenetic data, strain 15-13(T) represents a novel species of the genus Halomonas, for which the name Halomonas alkalitolerans sp. nov. is proposed. The type strain for this novel species is 15-13(T) (=CGMCC 1.9129(T) =NBRC 106539(T)).

Heterologous expression of transaldolase gene Tal from Saccharomyces cerevisiae in Fusarium oxysporum for enhanced bioethanol production.

The filamentous fungus Fusarium oxysporum is known for its ability to ferment xylose-producing ethanol. However, efficiency of xylose utilization and ethanol yield was low. In this study, the transaldolase gene from Saccharomyces cerevisiae has been successfully expressed in F. oxysporum by an Agrobacterium tumefaciens-mediated transformation method. The enzymatic activity of the recombinant fungus (cs28pCAM-Sctal4) was 0.195 times higher than that of the wild-type strain (cs28). The recombinant strain also exhibited a 28.83% increase in ethanol yield on xylose media compared to the parental strain. Enhanced ethanol production and a reduction in the biomass were observed during xylose fermentation. Ethanol yield from rice straw by simultaneous saccharification and fermentation with cs28pCAM-Sctal4 was 0.25 g g⁻¹ of rice straw. The transgenic strain of F. oxysporum cs28pCAM-Sctal4 might therefore have potential applications in industrial bioenergy production.

The characterization of transaldolase gene tal from Pichia stipitis and its heterologous expression in Fusarium oxysporum.

The 972 bp length of transaldolase gene tal was cloned from Pichia stipitis CICC1960, encoding a 323 amino acid protein with a calculated molecular mass of 35.36 kDa and isoelectric point of 5.20. Real time PCR analysis demonstrated that the mRNA transcript level of constitutive tal gene rise on xylose, glucose, fructose, mannose, galactose and sucrose as carbon source, respectively. Furthermore, the transcription of tal gene in P. stipitis on xylose was higher than on other carbon source, indicating that transaldolase plays a part in xylose utilization. To deeply study the tal gene biological function, it was expressed in Fusarium oxysporum CCTCC M209040. Recombinant transaldolase activity of transformant F. oxysporum M209040-Tal2 was about 0.52 U mg(-1) protein and was 1.57 times higher than that of the wild type F. oxysporum CCTCC M209040, indicating that the improvement of transaldolase activity in transformant was due to expression of the exogenous tal gene. Growth of transformant F. oxysporum M209040-Tal2 without selection pressure did not affect the level of hygromycin resistance of the transformants, suggesting that integrated tal gene was stable in mitosis. Fermentation trials of F. oxysporum M209040-Tal2 showed that the ethanol yield improved by 8.39 and 11.71% on glucose and xylose substrates, respectively, demonstrating that the expression of tal gene from P. stipitis CICC1960 in F. oxysporum CCTCC M209040 could improve ethanol production.

Cloning of a heat-stable chitin deacetylase gene from Aspergillus nidulans and its functional expression in Escherichia coli.

A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated an activity of 0.77 U ml(-1) for the glycol chitin substrates, and its specific activity was 4.17 U mg(-1) for it. The optimal temperature and pH of the purified enzyme were 50 degrees C and 8.0, respectively. When glycol chitin was used as the substrate, K (m) was 4.92 mg ml(-1), and K (cat) showed 6.25 s(-1), thus the ratio of K (cat) and K (m) was 1.27 ml s(-1) mg(-1). The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.

Haloterrigena daqingensis sp. nov., an extremely haloalkaliphilic archaeon isolated from a saline-alkaline soil.

A haloalkaliphilic archaeon, strain JX313(T), was isolated from a saline-alkaline soil from Daqing, Heilongjiang Province, China. Its morphological, physiological and biochemical features and 16S rRNA gene sequence were determined. Colonies of the strain were orange-red and cells were non-motile cocci and Gram-stain-variable. The strain required at least 1.7 M NaCl for growth, with optimal growth occurring in 2.0-2.5 M NaCl. Growth was observed at 20-50°C and pH 8.0-10.5, with optimal growth at 35°C and pH 10.0. The G+C content of its genomic DNA was 59.3 mol%. Phylogenetic analysis of 16S rRNA gene sequences showed that strain JX313(T) is associated with the genera Haloterrigena and Natrinema and is most closely related to Haloterrigena salina XH-65(T) (96.2  % sequence similarity) and Haloterrigena hispanica FP1(T) (96.2 %). DNA-DNA hybridization experiments revealed that the relatedness of strain JX313(T) to type strains of related species of the genus Haloterrigena or Natrinema was less than 50 %. Furthermore, the cellular polar lipids of strain JX313(T), identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and mannose-2,6-disulfate (1→2)-glucose glycerol diether (S2-DGD), were consistent with the polar lipid characteristics of the genus Haloterrigena. Therefore, phylogenetic analysis, phenotypic assessment and chemotaxonomic data showed that JX313(T) represents a novel species within the genus Haloterrigena, for which the name Haloterrigena daqingensis sp. nov. is proposed. The type strain is JX313(T) (=CGMCC 1.8909(T) =NBRC 105739(T)).

Cloning and heterologous expression of a novel endoglucanase gene egVIII from Trichoderma viride in Saccharomyces cerevisiae.

Endoglucanase is a major cellulolytic enzyme produced by the fungus Trichoderma viride. The 1,317 bp cDNA of endoglucanase gene egVIII was cloned from T. viride AS3.3711, encoding a 438 amino acid protein with a calculated molecular mass of 46.86 kDa and isoelectric point of 4.32. Sequence analysis suggested that EGVIII belonged to the glycosyl hydrolase family 5. The N-terminal region of EGVIII contains a signal peptide sequence of 19 amino acid residues, indicating that it is an extracellular enzyme. Transcription of the egVIII gene in T. viride AS3.3711 can be induced by carboxymethyl cellulose sodium (CMC-Na), sucrose, microcrystalline cellulose, and corn stalk, and inhibited by glucose and fructose. The alpha-mating factor signal can effectively enhance the secretion of the recombinant EGVIII in Saccharomyces cerevisiae, as demonstrated by the enzymatic activity of recombinant yeast IpYEMalpha-xegVIII in the supernatant, which was 0.86 times higher than that of the IpYES2-egVIII. Recombinant endoglucanase EGVIII showed optimal activity at a temperature of 60 degrees C and pH of 6.0. It was stable when incubated from 35 degrees C to 70 degrees C for 1 h. The enzymatic activity of recombinant EGVIII was stable at a pH 3.0 to 7.5 at 50 degrees C and reached the highest level at 0.174U when activated by 75 mM of Zn(2+). The Michaelis-Menten constant (Km) and Kcat values for CMC-Na and cellotriose hydrolysis were 3.82 mg/ml, 9.56 s(-1) and 1.75 mg/ml, 7.08 s(-1), respectively. Transgenic yeast strain IpYEMalpha-xegVIII might be useful for renewable fuels industries.

[Cloning and expression of the endo-beta-glucanase III cDNA gene from Trichoderma viride AS3.3711].

To study the construction of yeast bioengineering strain which can degrade cellulosic waste, an endo-beta-glucanase III (EG III) cDNA gene of Trichoderma viride AS3.3711 was isolated with RT-PCR protocol. After sequencing it was constructed on S. cerevisiae induceable expression vector pYES2. A L9 (3(4)) orthogonal design was used to optimize yeast sonication assistant transformation. The expression of EG III gene was induced by 2% beta-D-glactose, the transcription and expression of it was detected by Northern blotting and Congo Red method respectively. The endo-beta-glucanase activity was assayed as CMCase activity with CMC-Na as a substrate. The results show that the ORF of EG III was 1254 bp, encoding 418 aa, deducing molecular weight 44.1 x 10(3), group 5 (sonication treat time 60 s, incubate 40 min, SS-DNA 150 microg, heat shock 5 min) was the optimum one of the orthogonal experiment, and EG III transformants can produced clear hydrolysis halos on the Congo-Red-CMC plate. The measure of the enzyme activity show that the expression product can be expressed in active forms and secreted to the medium. The enzyme activity was approached the highest level (0.041 U/mL) when the culture time was 60 h. The optimized enzyme reaction temperature was 50 degrees C and the optimized pH was 5.8.