Expanding the Genetic Toolkit of Extremely Thermophilic, Lignocellulose Degrading Caldicellulosiruptor bescii

Abstract

Caldicellulosiruptor bescii is an anaerobic extreme thermophile with optimal growth temperatures between 70-78 °C. As a lignocellulose-degrading bacteria, C. bescii shows promise as a host organism for transforming lignocellulosic feedstocks into biofuels, producing a more sustainable source of liquid fuels. In recent years, C. bescii has been genetically modified to produce final products such as alcohols and carboxylic acids. Currently, genetic modification of C. bescii is slow and labor intensive, making it difficult to perform the genetic manipulations necessary for fine tuning the metabolic pathways of this organism for industrial scale processes. Moreover, C. bescii’s genetic toolkit is lacking characterization of native promoters with varied expression levels for doing this metabolic engineering. This project works towards introducing thermophilic CRISPR-Cas9 technology into Caldicellulosiruptor to manipulate its genome more effectively and explores the use of CagFbFP as an anaerobic fluorescent reporter for promoter strength. Our work to date has focused on knocking-in the following thermophilic Cas9 variants into C. bescii: GeoCas9, AceCas9, CaldoCas9, ThermoCas9, and IgnaviCas9. In this proof of concept, each Cas9 will knock-out the restriction enzyme CbeI in C. bescii as it is integrated into the genome. Then a replicating plasmid containing guide RNA (sgRNA) and regions for homology directed repair will be used to direct Cas9 to cut and modify the lactate dehydrogenase (ldh) gene in C. bescii. This will eliminate lactate production in the mutant strain; lactate production can then be measured to determine the efficiency of each Cas9 as a genome editing tool.