Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation.

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. KEY POINTS: • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase.

Chemical Stability and Characterization of Degradation Products of Blends of 1-(2-Hydroxyethyl)pyrrolidine and 3-Amino-1-propanol.

Aqueous amine solvents are used to capture CO2 from various flue gas sources. In this work, the chemical stability of a blend of 3-amino-1-propanol (3A1P) and 1-(2-hydroxyethyl)pyrrolidine [1-(2HE)PRLD] was studied. The chemical stability tests were conducted both in batch and cycled systems using various oxygen and NOx concentrations, additives (iron), and temperatures. In the thermal degradation experiments with CO2 present, the blend was more stable than the primary amines [(3A1P or monoethanolamine (MEA)] but less stable than the tertiary amine 1-(2HE)PRLD alone. Similar stability was observed between MEA, 3A1P, and the blend in the batch experiments at medium oxygen concentration (21% O2) and no iron present. 1-(2HE)PRLD was more stable. However, the presence of high oxygen concentration (96% O2) and iron reduced the stability of 1-(2HE)PRLD significantly. Furthermore, in the case of the blend, the chemical stability increased with increasing promoter concentration in batch experiments. During the cyclic experiment, the amine loss for the blend was similar to what was previously observed for MEA (30 wt %) under the same conditions. A thorough mapping of degradation compounds in the solvent and condensate samples resulted in the identification and quantification of 30 degradation compounds. The major components in batch and cycled experiments varied somewhat, as expected. In the cyclic experiments, the major components were ammonia, 3-(methylamino)-1-propanol (methyl-AP), N,N'-bis(3-hydroxypropyl)-urea (AP-urea), pyrrolidine, formic acid (formate), and N-(3-hydroxypropyl)-glycine (HPGly). Finally, in this paper, formation pathways for the eight degradation compounds (1,3-oxazinan-2-one, AP-urea, 3-[(3-aminopropyl)amino]-1-propanol, tetrahydro-1-(3-hydroxypropyl)-2(1H)-pyrimidinone, methyl-AP, N-(3-hydroxypropyl)-formamide, N-(3-hydroxypropyl)-ß-alanine, and HPGly) are suggested.

A comparative biodistribution study of polymeric and lipid-based nanoparticles.

Biodistribution of nanoencapsulated bioactive compounds is primarily determined by the size, shape, chemical composition and surface properties of the encapsulating nanoparticle, and, thus, less dependent on the physicochemical properties of the active pharmaceutical ingredient encapsulated. In the current work, we aimed to investigate the impact of formulation type on biodistribution profile for two clinically relevant nanoformulations. We performed a comparative study of biodistribution in healthy rats at several dose levels and durations up to 14-day post-injection. The studied nanoformulations were nanostructured lipid carriers incorporating the fluorescent dye IR780-oleyl, and polymeric nanoparticles containing the anticancer agent cabazitaxel. The biodistribution was approximated by quantification of the cargo in blood and relevant organs. Several clear and systematic differences in biodistribution were observed, with the most pronounced being a much higher (more than 50-fold) measured concentration ratio between cabazitaxel in all organs vs. blood, as compared to IR780-oleyl. Normalized dose linearity largely showed opposite trends between the two compounds after injection. Cabazitaxel showed a higher brain accumulation than IR780-oleyl with increasing dose injected. Interestingly, cabazitaxel showed a notable and prolonged accumulation in lung tissue compared to other organs. The latter observations could warrant further studies towards a possible therapeutic indication within lung and conceivably brain cancer for nanoformulations of this highly antineoplastic compound, for which off-target toxicity is currently dose-limiting in the clinic.