Modular protein engineering:design and development of artificial cellulosomes multi-enzymatic complexes: novel strategies applied to cellulose hydrolysis

GOROJOVSKY, N; SANTOS, J; CRAIG, PO; GARRONE N; GOLDBAUM FA.; IGLESIAS RANDON, MR; ZYLBERMAN V

Virtual

Conferencia; The 1st International BioDesign Research Conference.; 2020

Oranizado por Stanford University, the University of Warwick, and BioDesign Research (BDR).

The enzymatic degradation of lignocellulose generates sugars that upon fermentation produce bioethanol. The enzymes currently used in this process are expensive and have low efficiency. It is economically relevant to increase their activity and stability.Cellulosomes are multienzymatic complexes that colocalize different cellulolytic enzymes and cellulose binding domains, increasing their degradation activity through enzymatic proximity and substrate targeting effects. However, the industrial production of natural cellulosomes has serious limitations because of the properties of their scaffolding protein. Our goal is to use the structure of an oligomeric protein that is highly stable and highly expressed in bacteria, as a scaffold for the colocalization of a consortium of cellulolytic enzymes and the development of artificial cellulosomes. For the assembly of these multienzymatic particles we use a non-covalent coupling strategy trough high affinity heterodimeric peptides complementary fused to the oligomeric scaffold and the target enzymes. For this purpose we cloned and expressed in E.Coli the catalytic domain of a variety of enzymes, including endoglucanases, exoglucanases, xylanases, beta glucosidases and cellulose binding domains from different organisms, fused to the coupling peptide. The amount and solubility of these fusion proteins were compared to the original isolated domains. The coupling peptide doesn?t perturb the solubility of the target proteins. At least one member of each functional category is solubly expressed in significant amounts and has been able to be purified by affinity chromatography. We present advances in their functional characterization.We have successfully assembled several of the target proteins to the oligomeric scaffold encouraging further development of the artificial cellulosomes. It is expected that these complexes will help to increase the enzymatic lignocellulose degradation, reducing the cost of bioethanol production.