Clostridium Acetobutylicum Minicellulosomes Display And Assembly On Pichia Pastoris Cell Surface

With the rapid growth of fossil fuels prices, the development of new sustainable greenalternative energy has become the primary task of the world; and the use of other non-foodcellulosic feedstocks to produce ethanol has become the hotspot in the fuel production. Thecellulases are the most important components for the efficient degradation of cellulose. Thecellulosome was found by the combination of biochemical, biophysical, immunochemical,and molecular biological techniques, and its concept has been defined and proofed. To date,the cellulosome researches are constantly developing.Clostridium acetobutylicum is widely used for liquid fuel production. Many researcherspursued to engineer it for directly utilizing cellulose to produce acetone-butanol-ethanol. ThePichia expression system and surface display systems have been widely used in biologicalresearch, such as protein expression, antibody production, whole-cell catalysis, biosensors,and enzyme immobilization. We aimed to explore whether the scaffolding protein CipA of C.acetobutylicum could be transferred and work in yeast and whether the NtEG and the E4catalytic domain might operate in cellulosomes when fused with a dockerin tag. Weconstructed a set of prokaryotic and eukaryotic expression vectors for studying thecohesin-dockerin interaction between the CipA and the E4SD or the NtEGD. Besidescomparing the enzyme activities of cellulases between in the free-state and in the incorporatedminicellulosomes, we compared the designed minicellulosomes in the anchored state with theminicellulosomes in the unanchored state. The main results of this study were described asfollows:Firstly, the expression vectors of the scaffolding protein CipA and the E4SD have beenconstructed, then transformed into E. coli BL21(DE3), respectively. The recombinantproteins were expressed in E coli, and purified by affinity chromatography. The cellulosomeswere assembled with the purified proteins CipA and E4SD. The PAGE and SDS-PAGEanalysis shown that the cellulosomes were assembled successfully. Compared with thefree-state E4SD, the hydrolysis efficiencies of E4SD in the unanchored stateminicellulosomes for carboxymethyl cellulose and microcrystalline cellulose degradation were enhanced up to levels of approximately1.3-fold and3.0-fold, respectively.Secondly, the NtEGD had been modeled by protein3D structure simulation tools andassessed by evaluation tools, then have been genetically engineered and transformed intoPichia pastoris X33, obtained the transformant X33-ND. The SDS-PAGE and enzymaticactivity measurements of the X33-ND supernatant shown that the fusion protein NtEGD hasbeen expressed in Pichia pastoris.Thirdly, the yeast Pichia pastoris was genetically engineered to assembleminicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldinCipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysisconfirmed that CipA was targeted to the yeast cell surface and that NtEGD interacted withCipA on the yeast cell surface, suggesting that the cohesin and dockerin domains andcellulose-binding module (CBM) of C. acetobutylicum were functional in the yeasts. Theenzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeastcell surfaces increased dramatically. Additionally, the hydrolysis efficiencies of NtEGD incellulosomes for carboxymethyl cellulose, microcrystal cellulose and filter paper increased upto1.4-fold,2.0-fold and3.2-fold, respectively. The hydrolysis efficiencies of E4SD inanchored state cellulosomes for the carboxymethyl cellulose, microcrystal cellulose and filterpaper were increased up to3.5-fold,4.7-fold and3.0-fold, respectively. When β-glucosidasewas added to the reaction system, the hydrolytic activities of the free-state NtEGD enzymesand the minicellulosomes’ NtEGD both in the unanchored and anchored state proportionallyincreased for CMC and MC hydrolysis, while significant enhancement was also observedduring filter paper hydrolysis.Our results demonstrated that the truncated scaffoldin of C. acetobutylicum can beexpressed in E. coli and on the yeast cell surfaces, and assembled minicellulosomes with otherspecies’ cellulases by cohesin-dockerin interaction. These result means that like otherclostridial cellulosomes, the C. acetobutylicum cellulosome can also be expressed andassembled in the yeast. In addition, the incorporation of the endo-β-1,4-glucanases NtEG andE4into surface-displayed minicellulosomes demonstrated that this strategy allowed theadvantageous cellulases with favorable catalytic activities from different distant species couldbe incorporated into designed cellulosomes by an appended dockerin tag. These findings provide a novel insight into the process of cellulosome assembly. Potentially, thesurface-displayed cellulosomes that were described in this study may be utilized to develop anovel approach for the engineering of S. cerevisiae to produce ethanol from cellulose. Theymay also promote the development of further novel approaches in yeast engineering.