  2.4.1.B64 | more |
reverse engineering of a strain of Escherichia coli previously evolved for increased tolerance of octanoic acid (C8), an attractive biorenewable chemical, resulting in increased C8 production, increased butanol tolerance, and altered membrane properties. Evolution is determined to have occurred first through the restoration of WaaG activity, involved in the production of lipopolysaccharides, then an amino acid change in RpoC, a subunit of RNA polymerase, and finally mutation of the BasS-BasR two component system. The WaaG and RpoC mutations both contribute to increased C8 titers, with the RpoC mutation appearing to be the major driver of this effect. Each of these mutations contributes to changes in the cell membrane. Increased membrane integrity and rigidity and decreased abundance of extracellular polymeric substances can be attributed to the restoration of WaaG. The restoration of waaG occurrs first, and relatively quickly, with only the ML115 version of waaG being observed at the end of the second transfer and only the restored version of waaG (waaGR) being observed at the end of the third transfer. The parent strain with restored WaaG (strain YC005) results in a growth rate and final OD. The evolved strain phenotype can be completely attributed to waaGR and rpoCH419P, the basR mutation is not required |
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