S64: Dissection of the genome of an Escherichia coli strain genetically engineered and metabolically evolved to overproduce succinic acid

Myriant Corporation is constructing a plant in Louisiana to commercially produce succinic acid by fermentation from renewable carbon sources.  The Myriant process uses a series of Escherichia coli strains initially developed at the University of Florida (Jantama, et al., 2008. Biotechnol. Bioeng. 101:881-893).  Myriant has performed further work on the strains and fermentation methods to improve and better understand the process.

            Strain development was accomplished by both genetic engineering and metabolic evolution.  Deletion of unwanted anaerobic pathways to ethanol, lactate, formate and acetate resulted in strains that could only grow slowly by fermenting glucose to succinate.  Metabolic evolution in small fermentors was then used to select for spontaneous mutants that grew faster but maintained succinate production from glucose as their primary route for carbon metabolism, resulting in commercially attractive strains.

            Genome sequencing revealed that more than twenty point and frameshift mutations, and two significant deletions, occurred in the chromosome during evolution.  We reinstalled relevant wild type alleles, one at a time, to determine the quantitative effect on growth and succinate production.  In almost all cases, the wild type allele led to at least a small decrease in growth and/or succinate production.   Thus the desirable properties had resulted from the summation of many distinct mutations.  Knowledge of these mutations provides deep insight into how the strains are capable of producing commercially attractive levels of succinate. 

            Acknowledgment:   Material based in part on work supported by Department of Energy Award DE-EE0002878/001.  Disclaimer language prescribed in the award is incorporated herein in its entirety.