Stephen Van Dien and Anthony Burgard. Genomatica, Inc., 10520 Wateridge Circle, San Diego, CA 92121
Use of in silico modeling technologies to guide rational strain engineering can greatly accelerate the pace of industrial bioprocess development. One such technique is the OptKnock approach, which identifies a set of gene deletions designed to couple product formation to growth; i.e., in order for the cell to grow efficiently, it must also produce the compound of interest. A complementary experimental approach, evolutionary engineering, uses controlled selection pressure to optimize strain fitness and growth rate following genetic manipulations. In addition to achieving superior product yield, strains generated by this approach are suitable for continuous bioprocessing, due to their inherent genetic stability. OptKnock was used to design two E. coli strains for the growth-coupled production of succinic acid, a promising biorefinery product due to its vast array of potential derivatives including 1,4-butanediol. The first strain, AB3, contained 3 gene deletions and had anaerobic succinate yield on glucose of 21 mole %, approximately 5 times that of wild-type E. coli. The second, AB6, contained 6 gene deletions and produced a yield of 72 mole%. To increase the rates of both product formation and growth, these strains were then subjected to adaptive evolution using a specially designed machine to provide frequent serial dilutions. For example, 30 days of evolution increased product yield of AB3 to 113 mole%, which is very close to the maximum predicted for the strain. The strains were finally tested in a continuous fermentation to demonstrate superior productivity as well as genetic stability.
