Background: FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses. Results: Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively. Conclusions: Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

The aim was to investigate the effects of the cold dehulling of buckwheat seeds on their germination, total phenolic content (TPC), antioxidant activity (AA) and phenolics composition. Cold dehulling had no negative effects on germination rate and resulted in faster rootlet growth compared to hulled seeds. Although the dehulling of the seeds significantly decreased TPC and AA, the germination of dehulled seeds resulted in 1.8-fold and 1.9-fold higher TPC and AA compared to hulled seeds. Liquid chromatography coupled to mass spectrometry identified several phenolic compounds in free and bound forms. Rutin was the major compound in hulled seeds (98 µg/g dry weight), orientin and vitexin in 96-h germinated dehulled seeds (2205, 1869 µg/g dry weight, respectively). During germination, the increases in the major phenolic compounds were around two orders of magnitude, which were greater than the increases for TPC and AA. As well as orientin and vitexin, high levels of other phenolic compounds were detected for dehulled germinated seeds (e.g., isoorientin, rutin; 1402, 967 µg/g dry weight, respectively). These data show that dehulled germinated seeds of buckwheat have great potential for use in functional foods as a dietary source of phenolic compounds with health benefits.

Quercetin is a flavonoid that occurs in many types of fruit and vegetables and is stable for no longer than 4.5 h in the investigated pH range (6.0-8.0), even at 4 °C in the dark. At higher temperatures, the degradation/oxidation process is much faster. Simple but effective proliposomal encapsulation was used to protect the quercetin from environmental conditions such as pH. With this approach, 65 to 90% of pure quercetin and quercetin-rich onion extract was kept after >60 days under conditions that favoured its oxidation (pH 7.4). In addition, the encapsulated quercetin decreases the lipid peroxidation induced by pulsed UV light by >50%. At a mass ratio of 1:100 quercetin to lipids (w/w), the liposomes remained intact in solutions for six months. Quercetin in lipid bilayers simultaneously protects the unsaturated lipids from peroxidation.