Investigation of Novel Cellulose-Binding Tapirin Proteins in Caldicellulosiruptor Species

In traditional bioprocessing systems for the production of biofuels, plant biomass must undergo thermal/chemical pretreatment and enzymatic degradation before sugars are readily available and fermented into alcohols. By utilizing bacteria from the extremely thermophilic genus Caldicellulosiruptor, these multiple steps can be combined into one step for consolidated bioprocessing (CBP).  Key novel proteins, named tāpirins, were identified in these microorganisms and shown to be capable of binding to insoluble lignocellulosic biomass. To investigate these proteins further, two tāpirins were deleted (individually and simultaneously) from the genome of Caldicellulosiruptor bescii.  These knockout strains were created through multiple crossovers obtained by selecting for or against uracil auxotrophy and/or high temperature kanamycin resistance.   Further testing of the knockout strains in C. bescii will provide information on the strains’ ability to solubilize lignocellulose, specifically switchgrass and Avicel (crystallized cellulose).  Another tāpirin, Calkro_0844 from Caldicellulosiruptor kronotskyensis, is being expressed in Escherichia coli using Gibson Assembly to construct the desired vector with the tāpirin insert.  A C-terminal fragment of the Calkro_0844 protein has been crystallized and the binding domain identified; however, the entire tāpirin protein will be crystallized in order to gain a more comprehensive view of how the protein functions.  The potential for these thermophilic organisms to bind and degrade biomass is promising and, with more extensive testing, a better understanding of these abilities can be achieved and utilized in bioprocessing.