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

Abstract

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.

Biosynthesis of rapamycin and its regulation: past achievements and recent progress

“…Therefore, further improvement of the rapamycin titer and generation of diverse derivatives of rapamycin in S. hygroscopicus has been imperative for its commercial development and clinical applications. 62 In an attempt to improve rapamycin productivity, fermentation medium optimization as described in the previous section, 16,18,53,54 in addition to classical strain improvement, 63 protoplasts-related techniques 64 and high-throughput screening method 65 have been developed. Demain's research group obtained a higher rapamycin-producing mutant from the protoplasts of S. hygroscopicus FC904 mutants treated with 1 mg ml À1 of gentamicin, widely used aminoglycosides, where an HPLC analysis showed that the rapamycin titer for this mutant (139 mg l À1 ) was 60% higher than that for the parent strain.…”

“…However, the combination of interspecies protoplasts fusion and one round of genome shuffling generated a high-yield rapamycin producer with an outstanding yield of 445 mg l À1 . 64 Furthermore, as a result of the protoplast-related mutation and fusion experiments, it was observed that nearly all the highyield rapamycin-producing strains shared a similar phenotype: round colonies with wrinkled edges and abundant yellow aerial mycelia.…”

Comparative metabolic profiling reveals the key role of amino acids metabolism in the rapamycin overproduction by Streptomyces hygroscopicus

“…With the widespread clinical application and promising prospects, rapamycin has recently attracted much attention of many researchers and pharmaceutical companies. So far, considerable efforts have been made to improve rapamycin production, such as fermentation technology optimization [7,26,27], traditional strains mutation [8,46,50], and protoplast-related techniques [5]. Notably, ultraviolet mutagenesis, as a traditional method, has been successfully applied in the field of microbial breeding.…”

Strain improvement in actinomycetes in the postgenomic era

“…More recently, mutagenesis and protoplast fusion have been combined for whole genome shuffling to improve the production of antibiotics [22,72,75]. In addition, a number of molecular engineering strategies, often focused on optimizing precursor levels or expression of rate-limiting enzymes involved in secondary metabolite biosynthesis, have been summarized in other reviews [3][4][5][6]51].…”