Yunliu Fan

An acidophilic β-galactosidase from Bispora sp. MEY-1 with high lactose hydrolytic activity under simulated gastric conditions.

BgalA, a full-length gene (3,009 bp) that encodes a beta-galactosidase, was cloned from the meso-acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. The deduced amino acid sequence of BgalA shares highest identity (55.5%) with the beta-galactosidase from Aspergillus phoenicis, which belongs to the glycoside hydrolyase family 35. Purified recombinant BgalA is acidophilic, exhibiting maximum activity at pH 1.5, which is lower than that reported for other beta-galactosidases. The enzyme has high pH and thermal stability and is resistant to proteases and cations found in milk. The K(m) and V(max) of BgalA for 2-nitrophenyl-beta-D-galactopyranoside and lactose are 5.22 mM and 120.8 micromol/(min x mg), and 0.31 mM and 137.3 micromol/(min x mg), respectively. Under simulated gastric conditions, BgalA has greater stability ( approximately 100%) and hydrolysis ratio (>80%) toward milk lactose than the commercially available beta-galactosidase from Aspergillus oryzae (ATCC 20423). Thus, BgalA may be a better digestive supplement for alleviating symptoms associated with lactase deficiency.

Identification of a highly efficient stationary phase promoter in Bacillus subtilis

A promoter that enabled high-level expression of the target gene during the stationary phase in the absence of an inducer would facilitate the efficient production of heterogeneous proteins at a low cost. In this study, a genome-scale microarray-based approach was employed to identify promoters that induced high-level expression of the target genes in Bacillus subtilis from the late log phase to the stationary phase without an inducer. Eleven candidate promoters were selected based on B. subtilis microarray data and the quantitative PCR analysis. Among the selected promoters, Pylb exhibited the highest activity with the reporter bgaB during the stationary phase. Compared with P43 (a commonly used constitutive promoter), promoter Pylb could express two reporter genes (egfp and mApple), and the expression levels of EGFP and RFP were 7.8- and 11.3-fold higher than that of P43, respectively. This finding was verified by overexpression of the genes encoding pullulanase and organophosphorus hydrolase, the activities of which were 7.4- and 2.3-fold higher, respectively, when driven by Pylb compared with P43. Therefore, our results suggest that the Pylb promoter could be used to overexpress target genes without an inducer; this method could facilitate the identification and evaluation of attractive promoters in the genome.

Improvement of the Thermostability of Xylanase by N-terminus Replacement

The hybrid xylanase TB was constructed by the substitution of the N-terminus segment of the Streptomyces olivaceoviridis xylanase XYNB with corresponding region of Thermomonosporafusca xylanase TfxA. The hybrid gene tb, encoding the TB, was correctly expressed in Escherichia coli BL21 and Pichia pastoris GS115. TB was purified and its enzymatic properties were determined. The results revealed that the optimal temperature and optimal pH of TB were at 70 degrees C and 6.0, which have been obviously improved compared with those of XYNB. The thermostability of TB were all about six-fold of XYNBs after incubating the properly diluted enzyme solutions at 80 degrees C and 90 degrees C for 3min, respectively. The pH stability of TB was 5 to approximately 9, which was narrower than that of XYNB. Still, TB remains a high specific activity as XYNB does. Analysis of a homology modeling and sequence similarity were used to reveal the factors influencing the enzymatic properties of TB and the discussion for the relationship between structure and function of xylanase was given.

Utility of Thermostable Xylanases of Mycothermus thermophilus in Generating Prebiotic Xylooligosaccharides

Xylooligosaccharides as emerging prebiotics are able to promote the growth of probiotic bacteria. In the present study, four neutral, thermostable xylanases (MtXyn11A, MtXyn11At, MtXyn11B, and MtXyn11C) from compost fungus Mycothermus thermophilus CGMCC3.18119 were overexpressed in Pichia pastoris GS115 and used to produce xylooligosaccharides from beechwood xylan. The enzymes showed similar enzymatic properties (maximal activities at pH 6.0-6.5 and 65 °C) but varied in catalytic efficiency and cleaving actions. MtXyn11A, MtXyn11At, and MtXyn11C mainly produced xylobiose (59-62%), xylose (16-20%), and xylotriose (16-19%), while MtXyn11B released xylobiose (51%), xylotriose (32%), and xylose (12%) as the main products. When using the xylan hydrolysates of different xylanases as the carbon source, four probiotic Lactobacillus strains Lactobacillus brevis 1.2028, Lactobacillus rhamnosus GG, Lactobacillus casei BL23, and Lactobacillus plantarum WCSF1 were confirmed to use the xylooligosaccharides efficiently (83.8-98.2%), with L. brevis 1.2028 as the greatest.

The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1.

Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/106 conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens.

Predicting synonymous codon usage and optimizing the heterologous gene for expression in E. coli

Of the 20 common amino acids, 18 are encoded by multiple synonymous codons. These synonymous codons are not redundant; in fact, all of codons contribute substantially to protein expression, structure and function. In this study, the codon usage pattern of genes in the E. coli was learned from the sequenced genomes of E. coli. A machine learning based method, Presyncodon was proposed to predict synonymous codon selection in E. coli based on the learned codon usage patterns of the residue in the context of the specific fragment. The predicting results indicate that Presycoden could be used to predict synonymous codon selection of the gene in the E. coli with the high accuracy. Two reporter genes (egfp and mApple) were designed with a combination of low- and high-frequency-usage codons by the method. The fluorescence intensity of eGFP and mApple expressed by the (egfp and mApple) designed by this method was about 2.3- or 1.7- folds greater than that from the genes with only high-frequency-usage codons in E. coli. Therefore, both low- and high-frequency-usage codons make positive contributions to the functional expression of the heterologous proteins. This method could be used to design synthetic genes for heterologous gene expression in biotechnology.

Six new soil–inhabiting Cladosporium species from plateaus in China

ABSTRACT Cladosporium species are ubiquitous in various environments but are hitherto rarely isolated from soil. In the present study, six new Cladosporium species inhabiting the plateau soils of China are described as C. neopsychrotolerans, C. paralimoniforme, C. prolongatum, C. sinuatum, C. tianshanense, and C. verruculosum. These species are phylogenetically distinct and morphologically different from known species. This study increased the number of species classified in the C. cladosporioides and C. herbarum complexes and revealed Chinese plateau soil as a rich niche of Cladosporium species diversity.

Insight into the cold adaptation and hemicellulose utilization of Cladosporium neopsychrotolerans from genome analysis and biochemical characterization

The occurrence of Cladosporium in cold ecosystems has been evidenced long before, and most of the knowledge about nutrient utilization of this genus is sporadic. An alpine soil isolate C. neopsychrotolerans SL-16, showing great cold tolerance and significant lignocellulose-degrading capability, was sequenced to form a 35.9 Mb genome that contains 13,456 predicted genes. Functional annotation on predicted genes revealed a wide array of proteins involved in the transport and metabolism of carbohydrate, protein and lipid. Large numbers of transmembrane proteins (967) and CAZymes (571) were identified, and those related to hemicellulose degradation was the most abundant. To undermine the hemicellulose (xyaln as the main component) utilization mechanism of SL-16, the mRNA levels of 23 xylanolytic enzymes were quantified, and representatives of three glycoside hydrolase families were functionally characterized. The enzymes showed similar neutral, cold active and thermolabile properties and synergistic action on xylan degradation (the synergy degree up to 15.32). Kinetic analysis and sequence and structure comparison with mesophilic and thermophilic homologues indicated that these cold-active enzymes employed different cold adaptation strategies to function well in cold environment. These similar and complementary advantages in cold adaptation and catalysis might explain the high efficiency of lignocellulose conversion observed in SL-16 under low temperatures.

Identification of an operon involved in fluoride resistance in Enterobacter cloacae FRM

Fluorine is ubiquitous and the most active non-metal element in nature. While many microorganisms have developed fluoride resistance as a result of the widespread and prolonged application of oral hygiene products, the mechanisms used by these organisms to overcome fluoride toxicity are incompletely understood. In this study, a fluoride-resistant strain, Enterobacter cloacae FRM, was identified which could grow well at a fluoride concentration of 4,000 mg/L. According to comparative genomics, transcriptome under fluoride stress, and sequence analyses of two fluoride-resistant fosmid clones, the genomic island GI3 was found to be important for fluoride resistance. The result of quantitative RT-PCR indicated that six genes on GI3, ppaC, uspA, eno, gpmA, crcB, and orf5249, which encode a fluoride transporter, fluoride-inhibited enzymes, and a universal stress protein, reside in an operon and are transcribed into two mRNAs activated by fluoride with a fluoride riboswitch. The results of knockout and complementation experiments indicated that these genes work together to provide high fluoride resistance to E. cloacae FRM. This study clarified the resistance mechanism of this high fluoride-resistant organism and has expanded our understanding of the biological effects of fluoride.