Generation of high-yield rapamycin-producing strains through protoplasts-related techniques.

Rapamycin is a 31-member ring macrolide produced by Streptomyces hygroscopicus and has many applications in clinical medicine. In the present work, several protoplasts-related techniques including protoplasts mutation, intraspecies and interspecies protoplasts fusion were tried to improve the rapamycin productivity in S. hygroscopicus. Although mutation and fusion of different protoplasts of S. hygroscopicus did not improve the productivity of rapamycin significantly, the interspecies fusion of protoplasts of S. hygroscopicus D7-804 and Streptomyces erythreus ZJU325 could have brought about one high-yield (345 mg/L) rapamycin producer with 23.6% higher than that of the parental strain. Then, with seven mutants of S. hygroscopicus with different features and rapamycin productivities as the parental strains, only one-round genome shuffling has generated a high-yield rapamycin-producing strain with an outstanding yield of 445 mg/L. The systematic research of protoplast-related techniques has established an applicable way to generate high-yield strains from original microorganisms which can only produce low amount of expected natural products, without information of target gene clusters and gene sequences.

A high-throughput method for screening of rapamycin-producing strains of Streptomyces hygroscopicus by cultivation in 96-well microtiter plates.

A novel high-throughput cultivation method was developed to rapidly screen large numbers of rapamycin-producing mutants of Streptomyces hygroscopicus by duplicate culturing of isolates on the surfaces of agar-solidified 96 wells in microtiter plates. One copy of the cultures was used for the rapamycin bioassay and the other identical copy, representing potentially high yielding strains, was preserved for further study. By integrating 96-well solid cultivation and the bioassay, we screened more than 7000 isolates and found 10 high-yielding strains. From these, one mutant produced 420 mug rapamycin/ml, which was double the yield of parent strain used in the submerged fermentation process.

Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy.

Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.