Sporolactobacillus pectinivorans sp. nov., an anaerobic bacterium isolated from spoiled jelly.

A Gram-positive-staining, endospore-forming, facultatively anaerobic, lactic-acid-producing bacterium, strain GD201205T, was isolated from spoiled jelly in China. Strain GD201205T fermented glucose, fructose, mannose, sucrose, raffinose and turanose, but negative for nitrate reduction, catalase and oxidase. The predominant fatty acids of the strain were anteiso-C17 : 0 and anteiso-C15 : 0. Whole-cell hydrolysates contained glycine and alanine with meso-iaminopimelic acid as the diagnostic diamino acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, glycolipid 1 and glycolipid 2. The DNA G+C content of strain GD201205T was 48.7 mol%. 16S rRNA gene sequence analysis indicated that the strain belonged to the genus Sporolactobacillus and was most closely related to Sporolactobacillus vineaeKCTC 5376T and Sporolactobacillus putidusJCM 15325T with 16S rRNA gene sequence similarities of 97.5 and 96.9 %, respectively. Levels of DNA-DNA relatedness between strain GD201205T and Sporolactobacillus vineaeKCTC 5376Tand Sporolactobacillus putidusJCM 15325T were 29.2 and 47.6 %, respectively. Phylogenetic analysis based on the 16S rRNA gene and gyrB gene revealed that strain GD201205T was clearly distinct from all related species of the genus Sporolactobacillus. On the basis of the phylogenetic, chemotaxonomic and phenotypic evidence given in this study, strain GD201205T should be classified as a representative of a novel species of the genus Sporolactobacillus for which the name Sporolactobacillus pectinivorans is proposed. The type strain is GD201205T (=CICC 23867T=KCTC 15488T).

[Optimization of the expression of human DNA topoisomerase I in Pichia pastoris].

Human DNA Topoisomerase I (hTopo I) has been identified to be an efficient target of many effective antitumor drugs. Natural hTopo I is not convenient to be used in screening because of its low concentration in cells. In order to fast screen new anticancer drugs targeting at hTopo I from natural compounds in vitro, hTopo I gene open reading frame (ORF) has been successfully cloned and overexpressed in Pichia pastoris. Total RNA extracted from Hela cells was reversely transcripted to synthesize cDNA with the hTopo I specific antisense primer and the hTopo I ORF was synthesized by PCR. After digestion with EcoR I and Kpn I, the synthesized fragment was inserted into pPICZaA, gave rise to pPICZalpha-hTopoI. After digestion with Sac I, the lined pPICZalpha-hTopoI was transformed into Pichia pastoris strains (KM71, X33 and SMD1168) by electroporation and integrated into their genome. After screened on YPDS plates (containing 1000 ug/mL zeocin), the high-copy recombinant strains (KM-hTopoI, X33-hTopoI and SMD-hTopol) could overexpress recombinant hTopo I, which was fused to the alpha-factor secretion signal and could be secreted into the supernatant in the culture. alpha-factor could be cleaved from the expressed protein during secretion. A higher activity amount of the enzyme was secreted by the particular strain SMD-hTopoI because of its absence of proteimase A than by other strains which possess proteinase A activity. After optimizing the fermentation conditions, a relatively higher enzyme activity in the culture supernatant could be obtained when SMD-hTopoI was induced in BMMY (pH7.25) at 20 degrees C , with addition of 0.5% (V/V) methanol and 3% (V/V) nutrient liquid every 24h. The enzyme activity reached 43 000 u/mL, the yield reached 11 mg/L, achieving approximate 10% of total protein in the culture supernatant. SDS-PAGE and Western blot analyses showed that the mass of the recombinant hTopo I was 91 kD with no glycosylation.