Citrobacter freundii as a test platform for recombinant cellulose degradation systems: Citrobacter freundii for biomass degradation

Cellulosic biomass represents a huge reservoir of renewable carbon, but converting it to useful products is challenging. Attempts to transfer cellulose degradation capability to industrially useful microorganisms have met with limited success, possibly due to poorly understood synergy between multiple cellulases. This is best studied by co-expression of many combinations of cellulases and associated proteins. Here we describe the development of a test platform based on Citrobacter freundii, a cellobiose-assimilating organism closely related to Escherichia coli. Standard E. coli cloning vectors worked well in C. freundii. Expression of cellulases CenA and Cex of Cellulomonas fimi in C. freundii gave recombinant strains which were able to grow at the expense of cellulosic filter paper or microcrystalline cellulose (Avicel) in a mineral medium supplemented with a small amount of yeast extract. Periodic physicalagitation of the cultures was highly beneficial for growth at the expense of filter paper. This provides a test platform for the expression of combinations of genes encoding biomass degrading enzymes to develop effective genetic cassettes for degradation of different biomass streams.