Generation of high rapamycin producing strain via rational metabolic pathway‐based mutagenesis and further titer improvement with fed‐batch bioprocess optimization

Abstract

Rapamycin 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. Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have rationally designed approaches to generate a rapamycin high producer strain of S. hygroscopicus HD-04-S. These have included alleviation of glucose repression, improved tolerance towards lysine and shikimic acid, and auxotrophy of tryptophan and phenylalanine through the application of stepwise UV mutagenesis. The resultant strain produced rapamycin at 450 mg/L in the shake flask scale. These fermentations were further scaled up in 120 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. The final titer of rapamycin was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor. This work highlights a high rapamycin producing strain derived by mutagenesis and subsequent screening, fermentation optimization of which has now made it feasible to produce rapamycin on an industrial scale by fermentation. The strategies developed here should also be applicable to titer improvement of other important microbial natural products on an industrial scale.

Kinetics of rapamycin production by Streptomyces hygroscopicus MTCC 4003

“…The low titer of this drug produced by the organism has now become a rate-limiting factor in further development and industrialization of this natural product (Zhu et al 2010). In the past decades, most efforts have focussed on rapamycin biosynthesis (Park et al 2010; Graziani 2009), its pharmaceutical activities (Prapagdee et al 2008; Park et al 2010; Weber et al 2005; Foroncewicz et al 2005; Nicoletti et al 2009), strain improvement (Zhu et al 2010; Xu et al 2005; Chen et al 2009) and optimization of physiological factors for better production of rapamycin (Zou and Li 2013; Chen et al 2008). In the literature, although there are many reports describing optimization of the media (Refaat and Abdel-Fatah 2008) and improvement of strain for better production of rapamycin (Xu et al 2005; Jung et al 2011), kinetic studies of growth and rapamycin production by S. hygroscopicus have not been satisfactorily done yet.…”

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

“…The detailed UV mutagenesis was performed according to the methods described by Zhu et al [50] with slight modifications. The single spore suspension of S. hygroscopicus ATCC 29253 was exposed to UV rays at a distance of 20 cm from UV lamp for 10, 20, 30, 50, 60, 80, and 100 s, subsequently incubated on the ice bath for 2 h. To avoid any photoreaction resulting in reverse mutation of strains, all procedures and exposures were carried out in a dark room.…”

“…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.…”

Mutagenesis breeding of high echinocandin B producing strain and further titer improvement with culture medium optimization

“…Strain improvement plays a central role in the commercial development of microbial fermentation processes [4,10,29,31]. Although the modern techniques of gene engineering and metabolic engineering are considered to be effective methods in the microorganism breeding [11,16,27], the application of these modern techniques frequently encounters serious impediments due to limited insight into the genetics, physiology, and biochemistry of organisms [21].…”