Targeted and evolutionary genetic approaches to optimizng ethanol production in Clostridium thermocellum

The switch from food crops to cellulosic plants in biofuel production requires organisms to convert plant material to fuels.  Clostridium thermocellum is an anaerobic, thermophilic microorganism capable of degrading plant cell walls and producing ethanol.  In order to optimize ethanol production in Clostridium thermocellum, every other major metabolic pathway was blocked by deleting ldh, pta-ack, pfl, and hydG.  This strategy minimized side product generation in order to increase carbon flux through the ethanol pathway. A 2.5-fold increase in ethanol production was seen during growth on 5 g/L crystalline cellulose - from 13 mM by the parent strain to 33 mM by the mutant.  Also, because of the decrease in organic acid production, the mutant strain was capable of utilizing higher loadings of Avicel while still generating ethanol.  Though the strain produced more ethanol, the growth rate and maximum optical density on cellobiose were far below that of the parent strain.  An evolutionary approach was taken, selecting for increased growth rate.  Strains were passaged for ~1500 generations, improving both growth rate and ethanol yield.  After evolution growth rate was comparable to the wild type strain, and 42 mM ethanol was produced on cellobiose.  These evolved populations were purified via single colony isolation.  A subset of single colony isolates was used for characterization and resequencing.  From the resequencing data, the genetic changes that occurred during evolution were identified by comparing to the unevolved mutant. These high-yield strains provide platforms for improving ethanol production further and insight into evolutionary strategies.