Engineering hyperthermostable designer cellulosomes

Over the past several decades, we have developed native cellulosome and artificial designer cellulosome complexes for efficient deconstruction of cellulosic biomass. The cellulosomes have been shown to be more effective than comparable free enzyme systems. More recently, microbial degradation of cellulose has been shown to proceed more efficiently at very high temperatures. The use of thermophilic and hyperthermophilic (>80°C) bacteria and their enzyme systems are thus favored for cellulose degradation over those of their mesophilic counterparts, but there are no known hyperthermophilic cellulosome-producing bacteria. In our quest to produce designer cellulosomes that would act at higher temperatures, we have employed various approaches, including directed evolution and consensus-guided mutagenesis of the enzyme components, as well as the use of enzymes from naturally hyperthermophilc bacteria. We have thus engineered cellulosomal scaffoldin subunits, which include cohesins from thermophilic bacteria, complexed with chimaeric dockerin-bearingenzymes derived from ahyperthermophilic bacterium Caldicellulosirupter bescii, thereby creating the first hyperthermostable designer cellulosome. Since the enzymes are glycosylated in the native bacterium, we produced the designer cellulosome components using C. bescii as a host cell system, which conferred an additional level of thermostability onto the resultant glycosylated designer cellulosome, which showed significantly higher synergistic activity among its enzymatic components. Nevertheless, the bottleneck in obtaining designer cellulosomes active at even higher temperatures was traced to the scaffoldin subunit, whose cohesins were derived from thermophilic (but not hyperthermophilic) bacteria. In order to circumvent this intrinsic barrier, we have applied novel technologies to covalently link the cohesin and dockerin modules, thereby precluding their disruption at high temperatures. The ensuing hyperthermostable designer cellulosomes were indeed active at temperatures above 80°C.