Improved production of Daptomycin in an airlift bioreactor by morphologically modified and immobilized cells of Streptomyces roseosporus

Abstract

The increased threat of drug resistance has challenged the existence of several conventional and non-conventional antibiotics in the recent times. Daptomycin is a novel cyclic-lipopeptide antibiotic produced by Streptomyces roseosporus that has progressed as a significant anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotic. But, the economic practicality of this highly valued secondary metabolite is deterred by its poor production and tedious processing methodology. The present study aims at strategic improvement of Daptomycin production through morphological variations of S. roseosporus cells. Free cells, pelletized cells and immobilized cells on ultra porous refractory brick and silk sachets were investigated for the production of Daptomycin in a lab-scale 2.0 l air-lift bioreactor. The effect(s) of nitrogen source, inoculum size and oxygen stress were analyzed for pellet formation of S. roseosporus. Interestingly, free cells produced 750 mg/l of Daptomycin in a single batch. But, the three phase broth viscosity increased due to vigorous growth of free cells which hampered the oxygen transfer rate. The cell degeneration over the time deterred pellet reusability. 1430 mg/l Daptomycin was produced using pellets for 2 batches. On the contrary, mechanical stability, buoyancy and reusability of refractory bricks and silk sachets were beneficial. Daptomycin production was recorded for 6–8 batches. Immobilized cells on refractory bricks and silk sachets led to 4895 mg/l and 3623 mg/l Daptomycin production respectively. Cell immobilization improved the three phase broth rheology and hence, the hydrodynamics within the reactor. Therefore, whole-cell immobilization could contribute to the ameliorated production of this life-saving drug.

Streptomycetes as platform for biotechnological production processes of drugs

“…In recent years, the classical strategies to control these parameters have been coupled with more innovative approaches like fermentation based on mycelium immobilization, control of the metabolic pathways and antibiotic precursor supplementation, strain genetic manipulation, or metabolic engineering techniques. In this perspective, the study of the morphology, performed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), or confocal-laser scanning fluorescence microscopy (CLSM), to correlate the mycelium changes with the different approaches employed and the quantity of secondary metabolites produced, has also resulted crucial in both submerged and immobilized cell cultivations (Pereira et al 2008 ; López-García et al 2014 ; Ng et al 2014 ; Chakravarty and Kundu 2016 ; Scaffaro et al 2017 ) (Fig. 2 ).…”

“…units·g cdw -1 ∙h -1 ) Wentzel et al 2012 S. roseosporus NRRL11379 Daptomycin Supplementation with sodium decanoate as antibiotic precursor and optimized fed-batch feeding with dextrose 11.0 g YE, 0.86 g Fe(NH 4 ) 2 SO 4 ·6H 2 O, 10.7 g dextrose, 72.0 g potato Dext, 7.2 g cane molasses 3.6-L fermentor, 30 °C, pH = 6.5, 3.5 vvm, 450 rpm, 288 h 0.81 0.003 Ng et al 2014 Streptomyces strain Secondary metabolite Fermentation strategies Fermentation medium (*1.0 L) Growth conditions Production (g·L -1 ) Productivity (g·L -1· h -1 ) Reference S. cyaneogrisueus ssp. noncyanogenus Nemadectin WHd + RT impeller configuration, 25–55% DO by O 2 supply, moderate agitation speed 20.0 g Glc, 90.0 g CS, 25.0 g SoyF, 5.0 g YE, 4.0 g CaCO 3 ,11.0 g MgSO 4 ·7H 2 O, 0.01 g CuSO 4 , 0.002 g CoCl 2 , 0.001 g MnSO 4 5.0-L fermentor, 28 °C, pH = 7.4, 1.3 vvm, 650 rpm,192 h 1.37 0.007 Song et al 2018 S. roseosporus NBRC 12910 Daptomycin Cell immobilization on ultra-porous refractory brick flakes or silk sachets, airlift bioreactor 10.0 g Glc, 30.0 g Dext, 20.0 g SoyF, 0.6 g Fe(NH 4 ) 2 SO 4 , 0.2 g KH 2 PO 4 2.0-L fermentor, 30 °C, pH = 7.0, 200 rpm, 132 h each cycle 4.89 on refractory bricks and 3.62 on silk sachets 0.005 (considering 8 cycles on refractory bricks and 6 cycles on silk sachets) Chakravarty and Kundu 2016 S. coelicolor M145 Actinorhodin Cell encapsulation in calcium alginate beads 20.0 g Glc, 0.2 g CSA, 10.0 g YE, 42.0 g MOPS, 0.5 g K 2 SO 4 , 20.2 g MgCl 2 Shake flask, 30 °C, pH = 6.8, 200 rpm, 192 h 3.17 0.016 López-García et al 2014 S. coelicolor M145 Actinorhodin and undecylprodigiosin ...…”

“…Supplementation with precursors coupled with medium optimization has been in the last years a wise strategy to improve the yield of antibiotics too. Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus and approved by the FDA in 2003 as a drug for skin infections, right-sided endocarditis, and bacteremia, as it is able to act against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci strains (Chakravarty and Kundu 2016 ). In S. roseosporus NRRL11379 cultivations, the sodium decanoate was employed as an effective precursor of daptomycin as it seems to enhance the expression of key proteins of the biosynthetic pathways like guanosine pentaphosphate synthetase, polyribonucleotide nucleotidyltransferase phosphorylase, and tripeptidylamino peptidase (Table 2 ) (Ng et al 2014 ).…”

“…Morphology is demonstrated to be critical for secondary metabolite production. The morphology of Streptomyces roseosporus , for example, has demonstrated to change according to the carbon and nitrogen source, the inoculum size, the air flow rate used, and the eventual addition of precursors (Ng et al 2014 ; Chakravarty and Kundu 2016 ). Because of its filamentous nature, S. roseosporus free cell or pellet fermentations could drive to low daptomycin production; during fermentation, a dense biomass network could be formed and that would increase the broth viscosity and limit the volumetric oxygen transfer coefficients (Chakravarty and Kundu 2016 ).…”

Rheological Characterization of Streptomyces roseosporus for the Production of Daptomycin

“…The dispersed mycelium network increased the shear stress and shear rate ratio. During the stationary phase, the morphology of S. roseosporus was substantially changed in submerged culture with glass beads, because the swollen hyphae in S. roseosporus were differentiated into arthrospores due to the rapid secretion of the antibiotic 16 . At low shear stress conditions (i.e.…”