Engineering Metabolism for Cellulosic Biofuel Production and Carbon Conservation

Biomass recalcitrance—resistance to degradation—currently limits the use of lignocellulose for biofuel production. Consolidated bioprocessing (CBP), in which cellulose hydrolysis and fermentation occur simultaneously in one pot without added cellulases, is a potential approach to improve lignocellulose utilization. Owing to its high cellulose deconstruction rate, Clostridium thermocellum is a promising thermophilic CBP host, which grows at 50-60oC. The elevated temperature also promotes cellulose degradation, reduces contamination, and minimizes cooling cost. The method for genetic manipulation of C. thermocellum has not been fully developed and remains time-consuming. To expedite the progress, our first Aim was to establish the desired metabolic pathways in a related, more tractable organism, Geobacillus thermoglucosidasius. This Aim was accomplished by establishing a thermophilic isobutanol pathway and demonstrating the feasibility of producing isobutanol from glucose and cellobiose at an elevated temperature using G. thermoglucosidasius. Our second Aim was then to directly produce isobutanol from cellulose using the CBP organism, C. thermocellum. To overcome the pathway toxicity and to accelerate the promoter selection process, we cloned the essential isobutanol pathway genes under different promoters to create various plasmid constructs for isobutanol production in C. thermocellum. We developed a Cre-lox based gene deletion protocol to facilitate chromosomal editing. We also characterized the electron flow in the isobutanol pathway and related pathways. Specifically, we identified the Por enzyme which converts pyruvate to acetyl-CoA for ethanol and acetate production. 9.7 g/L of isobutanol was produced by the engineered C. thermocellum strain directly from cellulose within 100 h.Intrinsic carbon lost from the Embden-Meyerhof-Parnas (EMP, known as glycolysis) pathway is another limitation for biofuel and biochemical production. Through glycolysis, sugar is converted to pyruvate, which is then decarboxylated to acetyl-CoA with CO2 formation. The carbon lost from glycolysis limits the theoretical carbon atom yield to 2/3. Recently, a synthetic non-oxidative glycolysis (NOG) pathway has been engineered in an Escherichia coli strain to conserve all carbon from xylose (Bogorad et al., 2013). However, the engineered strain still depended on EMP pathway for xylose catabolism. Our third Aim was to construct an E. coli strain that completely relies on NOG for sugar catabolism. Here we designed a rational/evolutionary strategy to allow organisms to grow based on NOG without EMP. This new type of E. coli strain could convert C6 or C5 sugar to C2 compound without carbon lost. At first, the EMP pathway and other potential bypass pathways were deleted in E. coli. Then the cells were evolved for growth on glucose with the supplementation of acetate. Then the NOG pathway was installed and the strain was evolved first in xylose and then in glucose for growth. Finally, the strain was used for producing more than 2 molecules of C2 compound (acetate) from one molecule of glucose