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Quantitative Detection of Methanotrophs in Soil by Novel pmoA-Targeted Real-Time PCR Assays. Steffen Kolb, 2003.Methane oxidation in soils is mostly accomplished by methanotrophic bacteria . Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome . Comparison of 16S ribosomal DNA and pmoA (  subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the
and
 subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils) . We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene . Detection limits were between 101 and 102 target molecules per reaction for all assays . Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria . Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs . Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 ± 1.4) x 106 pmoA molecules g-1 for all methanotrophs . The Methylosinus group was predominant (2.7 x 106 ± 1.1 x 106 target molecules g-1) . In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 106 ± 0.9 x 106 target molecules g of soil-1) . On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 104 target molecules g of soil-1 . Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil . This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature .
Enhanced Production of  -Galactosyl Epitopes by Metabolically Engineered Pichia pastoris.Jun Shao, 2003.A metabolically engineered Pichia pastoris strain was constructed that harbored three heterologous enzymes: an S11E mutated sucrose synthase from Vigna radiata, a truncated UDP-glucose C4 epimerase from Saccharomyces cerevisiae, and a truncated bovine -1,3-galactosyltransferase . Each gene has its own methanol-inducible alcohol oxidase 1 promoter and transcription terminator on the chromosomal DNA of P . pastoris strain GS115 . The proteins were coexpressed intracellularly under the induction of methanol . After permeabilization, the whole P . pastoris cells were used to synthesize
-galactosyl (-Gal) trisaccharide (Gal1,3Galß1,4Glc) with in situ regeneration of UDP-galactose . Up to 28 mM
-Gal was accumulated in a 200-ml reaction . The Pichia system described here is simple and flexible . This work demonstrates that recombinant P . pastoris is an excellent alternative to Escherichia coli transformants in large-scale synthesis of oligosaccharides .
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