High Efficient Production Of Rapamycin In Streptomyces Hygroscopicus With Strain Genetic Improvement And Subsequent Bioprocess Optimizations

Rapamycin (Sirolimus) is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications.We optimized the fermentation medium of the original high-yield mutant S. hygroscopicus with regard to the formation of rapamycin, and the optimal fermentation medium was formulated. The optimal medium:4% glucose,1% glycerol, 1% soybean meal,0.15% L-Lys,0.5% K2HPO4,0.5% KH2PO4,0.5% NaCl,0.2% MgSO4,0.15% FeSO4. Under these optimal fermentation conditions, shake bottle loading of 20ml/250ml, culture temperature of 28℃, and havest cell mass after cultivated for 5 days, the rapamycin production was improved to 120mg/L.Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have designed a rational screening approach to generate a rapamycin high producer strain. The breeding of the rapamycin producer was carried out by isolating high-producing strains from colonies after exposure to the mutagens of UV, NTG or/and DES. The resultant strain produced rapamycin at 279mg/L in the shake flask scale. Then 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. By integrating 96-well solid cultivation and the bioassay, we screened more than 100,000 isolates and one mutant produced 445mg rapamycin/L was screened out, which was double the yield of parent strain used in the submerged fermentation process.The fermentation processes were further scaled up in 120 L and 20,000 L fermentors, respectively at the pilot plant. Selected fermentation factors including agitation speed, pH, and on-line supplementation were systematically evaluated. A fed-batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. In the final fermentation titer, the rapamycin productivity was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor, respectively.Several protoplasts-related techniques including protoplasts mutation, intraspecies, and interspecies protoplasts fusion were carried out to improve the rapamycin productivity in S. hygroscopicus. The interspecies fusion of protoplasts of rapamycin producer S. hygroscopicus D7-804 and erythreumycin producer S. erythreus ZJU325 could have brought about one high-yield (345 mg/L) rapamycin producer with 23.6% higher than that of the parental strain. After genome shuffling work, two high-yielding strains were isolated and the highest productivity of rapamycin attained 445mg/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. Comparing with traditional screening method, genome shuffling is much rapid and higher efficiency to obtain higher productivity strain.The biosynthetic gene cluster of rapamycin(RAP) has been respectively integrated into three Streptomyces mode hosts S. albus, S. ceolicolor M512, and S. lividans K4-114 by BAC-based technology to generate the RAP heterologous production systems. The bioassay results suggested that olny S. albus 28-1-1 from the constructed three heterologous systems has clearly showed the antibiotic activity similar to RAP. But the parallel HPLC and LC-MS analyses did not detect any trace of the production. Subsequent RT-PCR analyses has indicated that the majority of RAP biosynthetic genes were not transcripted, only three downstream genes rapG, rapF and rapD showed transcription signal in S. albus. Thus, insufficient or improper regulation of RAP genes could be a critical reason that limited the normal gene transcription. This exploring study will be a base for efficient biosynthesis of rapamycin and its similarities in the future.