(459a) Engineering Escherichia coli strains for Optimal Performance in Co-Culture with Trichoderma Reesei for Consolidated Bioprocessing

Consolidated bioprocessing, in which enzyme production, hydrolysis, and fermentation occur in a single step, is predicted to offer the lowest cost configuration for cellulosic biofuel production given a sufficiently efficient process. Use of synthetic microbial consortia can help to address some of the limitations of monoculture bioprocessing, including high metabolic burden on the cells and the need to optimize multiple pathways or functions in the same species. Design of synthetic microbial consortia, however, brings new challenges including identifying or engineering strains that can perform optimally within a mixed population.

We are working on a synthetic co-culture of cellulase hyper-producer Trichoderma reesei strain RUT-C30 and Escherichia coli carrying a pathway for the drop-in biofuel isobutanol from glucose. This co-culture can produce isobutanol directly from cellulosic substrate in one consolidated bioprocess [1]. The E. coli strain NV3 [2] initially employed in this system performs suboptimally, achieving lower yield and titer, higher byproduct formation, and notably higher plasmid loss compared to monoculture conditions. Here we present our work to generate E. coli strains with superior co-culture performance. We have taken a directed evolution approach, selecting for E. coli strains with improved growth on valine analog norvaline in co-culture with T. reesei. Additionally we have integrated and optimized the copy number of the isobutanol production pathway genes in the E. coli genome to achieve a more stable process.

[1] Minty JJ, Singer ME, Scholz SA, Bae C-H, Ahn J-H, Foster CE, Liao JC, & Lin XN (2013) Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass. Proc Natl Acad Sci USA 110(36): 14592-14597.

[2] Smith KM & Liao JC (2011). An evolutionary strategy for isobutanol production strain development in Eschericia coli. Metab Eng 13(6): 674-81.