Ruminococcus flavefaciens 007C cellulosomes and cellulase consortium | Celulosomi in celulazni konzorcij Ruminococcus flavefaciens 007C

English. Ruminococcus flavefaciens is among the most important cellulolytic bacterial species in rumen and gastrointestinal tract of monogastric herbivorous animals. Its efficiency in degradation of (hemi)cellulosic substrates is associated with the production of remarkably intricate extracellular multienzyme complexes, named cellulosomes. In the present work we investigated the cellulolytic system of 007C. The bioinformatic analysis of the draft genome sequence revealed identical organization of sca gene cluster as has previously been found in four other strains of R. flavefaciens. The cluster consists of five genes in the following order: scaC-scaA-scaB-cttA-scaE. The cellulases of R. flavefaciens 007C belong to four families of glycoside hydrolases, namely GH48, GH44, GH9 in GH5. Majority of these enzymes are putative endoglucanases, belonging to families GH5 and GH9, whereas only one gene encoding GH44 and GH48 was found. Apart from catalytic domains, most of these proteins also contain dockerins – signature sequences, which indicate their attachement to cellulosomes. On the other hand, carbohydrate-binding modules were only found coupled to GH9 catalytic domains. Zymogram analysis showed that larger endoglucanases were mostly constitutively expressed, wheras smaller enzymes were only detected in later phases of Avicel-grown cultures.

Slovenian. Ruminococcus flavefaciens sodi med najpomembnejše celulolitične bakterijske vrste v vampu prežvekovalcev in debelem črevesu monogastričnih rastlinojedih sesalcev. Njena izjemna učinkovitost pri anaerobni razgradnji (hemi)celuloznih substratov je povezana s sintezo zunajceličnih multiencimskih kompleksov celulosomov. V pričujočem delu smo preučili genomski potencial za sintezo celulosomov in celulaz seva 007C, izražanje slednjih pa analizirali z encimogrami. V osnutku genoma tega seva smo identificirali v celoti ohranjeno gručo genov, ki kodirajo skafoldine, na osnovi njihove modularne zgradbe pa smo predvideli zgradbo celulosomov tega seva. Nabor genov za celulaze, ki smo jih identificirali v genomu seva 007C, obsega po en zapis za encima s katalitskima domenama GH48 in GH44 ter širok spekter domnevnih endoglukanaz iz družin GH9 in GH5. Pri večini teh encimov smo identificirali dokerinske domene, kar kaže na to, da so vezani v celulosome. Module za vezavo na celulozo smo identificirali le v kombinaciji s katalitskimi domenami GH9. Ugotovili smo tudi, da se večje endoglukanaze izražajo konstitutivno, manjše encime pa smo zaznali le v supernatantu kultur, ki so daljši čas rasle na mikrokristalinični celulozi.