Zhihua Jin

Genomic variability among high pristinamycin-producing recombinants of Streptomyces pristinaespiralis revealed by amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP) was used to analyze genomic variability between high pristinamycin-producing recombinants of Streptomyces pristinaespiralis produced by genome shuffling and their ancestral strain. The AFLP fingerprints obtained with two restriction enzyme combinations of ApaI/TaqI and PstI/SacII showed together that there was no major polymorphism (less than 10%) between these high yield recombinants and their ancestor. However, the unique polymorphic bands, which might be related to the yield increasing of pristinamycin, could be distinguished from all the recombinants. Clustering analysis further indicated that the recombinants with similar ability of pristinamycin production had similar genomic variability.

Effects of Glucose and Phosphate on Spinosad Fermentation by Saccharopolyspora spinosa

Abstract The effects of glucose and inorganic phosphate on mycelium growth and spinosad production with Saccharopolyspora spinosa were studied. The results showed that the increase of glucose concentration from 18.6g·L −1 to 58.8g·L −1 could promote the mycelium growth and spinosad production. And when the glucose concentration increased from 58.8g·L −1 to 79.6g·L −1 , no obvious change was detected but a slight drop in spinosad production was observed, whereas, when the glucose concentration increased from 79.6g·L −1 to 115.3g·L −1 , substantial decrease in both mycelium growth and spinosad production occurred. The increase of phosphate concentration from 3.68mmol·L −1 to 29.41mmol·L −1 rendered corresponding increase in mycelium growth and spinosad production. When phosphate concentration increased from 29.41mmol·L −1 to 44.12mmol·L −1 , mycelium growth slightly increased and spinosad production dropped, while when phosphate concentration increased from 44.12mmol·L −1 to 57.62mmol·L −1 , both mycelium growth and spinosad production decreased sharply. Conclusively, the optimal initial concentration of glucose and phosphate is 58.8g·L −1 and 29.41mmol·L −1 , respectively. The spinosad fermentation in the production medium containing 58.8g·L −1 glucose and 29.41mmol·L −1 phosphate was scaled up in 5-L fermentation and the spinosad production reached 507mg·L −1 , which was 28% higher than that in the flask fermentation.

Probing the Molecular Mechanisms for Pristinamycin Yield Enhancement in Streptomyces pristinaespiralis

The mechanisms for the enhancement of pristinamycin production in the high-yielding recombinants of Streptomyces pristinaespiralis obtained by genome shuffling were investigated by quantitative real-time PCR (Q-PCR) and amplified fragment length polymorphism (AFLP) techniques. Q-PCR analysis showed that snaB and snbA involved, respectively, in the biosynthesis of pristinamycins II and I component had more extended high expression in the recombinant than that in the ancestor during fermentation process, indicating their expression changes might be key factors during the biosynthesis of the antibiotic. In addition, the antecedent establishment of the high self-resistance to pristinamycin, because ptr resistance gene started high-level expression ahead of the onset of the antibiotic production in the recombinant, might also lead to the increase of the antibiotics yield. AFLP analysis of these recombinants revealed genome variation of two novel genes, the homologs of AfsR regulatory gene and transposase gene, indicating these two gene variations were probably responsible for yield improvement of pristinamycin. This study provided several potential molecular clues for pristinamycin yield enhancement.

Expanded Bed Recovery of D-2-Chloropropionic Acid Dehalogenase Using TiO2-Densified Cellulose Anion Exchanger

Abstract The TiO 2 -densified cellulose composite beads were activated by epichlorohydrin and coupled with diethylamine, to function as an anion exchanger for expanded bed chromatography. The adsorbent exhibited a favorable performance of expanded bed adsorption for proteins, and therefore was applied to the expanded bed recovery of D-2-chloropropionic acid dehalogenase directly from the unclarified homogenate of Pseudomonas sp. NT21. The binding capacity of the dehalogenase was found to be 8.54U·ml −1 adsorbent, and two active peaks were eluted respectively at 0.15mol·L −1 and 0.3mol·L −1 (NH 4 ) 2 SO 4 . The result indicated that the overall enzyme yield was 68%, with a purification factor of 22. In comparison to other recovery processes, the yield of the expanded bed process rises at least 70%, simultaneously saving a great deal of operation time and costs.